Polypeptides having cellobiohydrolase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having cellobiohydrolase activity, catalytic domains, and cellulose binding domains and polynucleotides encoding the polypeptides, catalytic domains, and cellulose binding domains. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides, catalytic domains, or cellulose binding domains.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/982,178filed Nov. 21, 2013, now U.S. Pat. No. 9,506,048, which is a 35 U.S.C.371 national application of PCT/US2012/22749 filed on Jan. 26, 2012,which claims priority or the benefit under 35 U.S.C. 119 of U.S.Provisional. Application No. 61/450,494 filed on Mar. 8, 2011, andEuropean Application No. 11152252.0 filed on Jan. 26, 2011, the contentsof which are fully incorporated herein by reference.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

This invention was made in part with Government support underCooperative Agreement DE-FC36-08G018080 awarded by the Department ofEnergy. The government has certain rights in this invention.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

This application contains a reference to a deposit of biologicalmaterial, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention relates to polypeptides having cellobiohydrolaseactivity, catalytic domains, and cellulose binding domains, andpolynucleotides encoding the polypeptides, catalytic domains, orcellulose binding domains. The invention also relates to nucleic acidconstructs, vectors, and host cells comprising the polynucleotides aswell as methods of producing and using the polypeptides, catalyticdomains, and cellulose binding domains.

Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently linked bybeta-1,4-bonds. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of lignocellulosic feedstocks into ethanol has theadvantages of the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the lignocellulose is converted tofermentable sugars, e.g., glucose, the fermentable sugars are easilyfermented by yeast into ethanol.

The present invention provides polypeptides having cellobiohydrolaseactivity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingcellobiohydrolase activity selected from the group consisting of:

(a) a polypeptide having at least 85% sequence identity to the maturepolypeptide of SEQ ID NO: 2;

(b) a polypeptide encoded by a polynucleotide that hybridizes under veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii);

(c) a polypeptide encoded by a polynucleotide having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or the cDNA sequence thereof;

(d) a variant of the mature polypeptide of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the polypeptide of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention also relates to isolated polypeptides comprising acatalytic domain selected from the group consisting of:

(a) a catalytic domain having at least 90% sequence identity to aminoacids 98 to 456 of SEQ ID NO: 2;

(b) a catalytic domain encoded by a polynucleotide that hybridizes undervery high stringency conditions with (i) nucleotides 397 to 1786 of SEQID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii);

(c) a catalytic domain encoded by a polynucleotide having at least 90%sequence identity to nucleotides 397 to 1786 of SEQ ID NO: 1 or the cDNAsequence thereof;

(d) a variant of amino acids 98 to 456 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the catalytic domain of (a), (b), (c), or (d) that hascellobiohydrolase activity.

The present invention also relates to isolated polypeptides comprising acellulose binding domain selected from the group consisting of:

(a) a cellulose binding domain having at least 90% sequence identity toamino acids 20 to 56 of SEQ ID NO: 2;

(b) a cellulose binding domain encoded by a polynucleotide thathybridizes under very high stringency conditions with (i) nucleotides 58to 273 of SEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) thefull-length complement of (i) or (ii);

(c) a cellulose binding domain encoded by a polynucleotide having atleast 90% sequence identity to nucleotides 58 to 273 of SEQ ID NO: 1 orthe cDNA sequence thereof;

(d) a variant of amino acids 20 to 56 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions; and

(e) a fragment of the cellulose binding domain of (a), (b), (c), or (d)that has cellulose binding activity.

The present invention also relates to isolated polynucleotides encodingthe polypeptides of the present invention; nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing the polypeptides.

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.

The present invention also relates to a polynucleotide encoding a signalpeptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2,which is operably linked to a gene encoding a protein; nucleic acidconstructs, expression vectors, and recombinant host cells comprisingthe polynucleotides; and methods of producing a protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of a Talaromyces byssochlamydoides Family GH6cellobiohydrolase in the hydrolysis of milled unwashed PCS at 50-65° C.by a high-temperature enzyme composition (HTM).

FIG. 2 shows a comparison of a Talaromyces byssochlamydoides GH6cellobiohydrolase (Tb6), Aspergillus fumigatus GH6A cellobiohydrolase(Af6A), and Myceliophthora thermophila GH6A cellobiohydrolase (Mt6A) onhydrolysis of milled washed PCS at 50-65° C. and pH 4.0-5.0 in thepresence of Aspergillus fumigatus beta-glucosidase.

DEFINITIONS

Acetylxylan esterase: The term “acetylxylan esterase” means acarboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetylgroups from polymeric xylan, acetylated xylose, acetylated glucose,alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of thepresent invention, acetylxylan esterase activity is determined using 0.5mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0containing 0.01% TWEEN™ 20 (polyoxyethylene sorbitan monolaurate). Oneunit of acetylxylan esterase is defined as the amount of enzyme capableof releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Alpha-L-arabinofuranosidase: The term “alpha-L-arabinofuranosidase”means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55)that catalyzes the hydrolysis of terminal non-reducingalpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzymeacts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.Alpha-L-arabinofuranosidase is also known as arabinosidase,alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase,polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranosidehydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of thepresent invention, alpha-L-arabinofuranosidase activity is determinedusing 5 mg of medium viscosity wheat arabinoxylan (MegazymeInternational Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100mM sodium acetate pH 5 in a total volume of 200 μl for 30 minutes at 40°C. followed by arabinose analysis by AMINEXO HPX-87H columnchromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

Alpha-glucuronidase: The term “alpha-glucuronidase” means analpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzesthe hydrolysis of an alpha-D-glucuronoside to D-glucuronate and analcohol. For purposes of the present invention, alpha-glucuronidaseactivity is determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of alpha-glucuronidase equals the amount of enzymecapable of releasing 1 μmole of glucuronic or 4-O-methylglucuronic acidper minute at pH 5, 40° C.

Beta-glucosidase: The term “beta-glucosidase” means a beta-D-glucosideglucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminalnon-reducing beta-D-glucose residues with the release of beta-D-glucose.For purposes of the present invention, beta-glucosidase activity isdetermined using p-nitrophenyl-beta-D-glucopyranoside as substrateaccording to the procedure of Venturi et al., 2002, Extracellularbeta-D-glucosidase from Chaetomium thermophilum var. coprophilum:production, purification and some biochemical properties, J. BasicMicrobiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0μmole of p-nitrophenolate anion produced per minute at 25° C., pH 4.8from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mMsodium citrate containing 0.01% TWEEN® 20.

Beta-xylosidase: The term “beta-xylosidase” means a beta-D-xylosidexylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of shortbeta (1→4)-xylooligosaccharides to remove successive D-xylose residuesfrom non-reducing termini. For purposes of the present invention, oneunit of beta-xylosidase is defined as 1.0 μmole of p-nitrophenolateanion produced per minute at 40° C., pH 5 from 1 mMp-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citratecontaining 0.01% TWEEN® 20.

Catalytic domain: The term “catalytic domain” means the region of anenzyme containing the catalytic machinery of the enzyme.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic or prokaryotic cell. cDNA lacks intron sequences thatmay be present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA that is processed through a seriesof steps, including splicing, before appearing as mature spliced mRNA.

Cellobiohydrolase: The term “cellobiohydrolase” means a1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176)that catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages incellulose, cellooligosaccharides, or any beta-1,4-linked glucosecontaining polymer, releasing cellobiose from the reducing ornon-reducing ends of the chain (Teen, 1997, Crystalline cellulosedegradation: New insight into the function of cellobiohydrolases, Trendsin Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reeseicellobiohydrolases: why so efficient on crystalline cellulose?, Biochem.Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determinedaccording to the procedures described by Lever et al., 1972, Anal.Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149:152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288;and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the presentinvention, the Tomme et al. method can be used to determinecellobiohydrolase activity.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, or at least 100% of the cellobiohydrolaseactivity of the mature polypeptide of SEQ ID NO: 2.

Cellulolytic enzyme or cellulase: The term “cellulolytic enzyme” or“cellulase” means one or more (e.g., several) enzymes that hydrolyze acellulosic material. Such enzymes include endoglucanase(s),cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. Thetwo basic approaches for measuring cellulolytic activity include: (1)measuring the total cellulolytic activity, and (2) measuring theindividual cellulolytic activities (endoglucanases, cellobiohydrolases,and beta-glucosidases) as reviewed in Zhang et al., Outlook forcellulase improvement: Screening and selection strategies, 2006,Biotechnology Advances 24: 452-481. Total cellulolytic activity isusually measured using insoluble substrates, including Whatman No 1filter paper, microcrystalline cellulose, bacterial cellulose, algalcellulose, cotton, pretreated lignocellulose, etc. The most common totalcellulolytic activity assay is the filter paper assay using Whatman No 1filter paper as the substrate. The assay was established by theInternational Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987,Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

For purposes of the present invention, cellulolytic enzyme activity isdetermined by measuring the increase in hydrolysis of a cellulosicmaterial by cellulolytic enzyme(s) under the following conditions: 1-50mg of cellulolytic enzyme protein/g of cellulose in PCS (or otherpretreated cellulosic material) for 3-7 days at a suitable temperature,e.g., 50° C., 55° C., or 60° C., compared to a control hydrolysiswithout addition of cellulolytic enzyme protein. Typical conditions are1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mMsodium acetate pH 5, 1 mM MnSO₄, 50° C., 55° C., or 60° C., 72 hours,sugar analysis by AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc.,Hercules, Calif., USA).

Cellulose binding domain: The term “cellulose binding domain” means theregion of an enzyme that mediates binding of the enzyme to amorphousregions of a cellulose substrate. The cellulose binding domain (CBD) istypically found either at the N-terminal or at the C-terminal extremityof an enzyme.

Cellulosic material: The term “cellulosic material” means any materialcontaining cellulose. The predominant polysaccharide in the primary cellwall of biomass is cellulose, the second most abundant is hemicellulose,and the third is pectin. The secondary cell wall, produced after thecell has stopped growing, also contains polysaccharides and isstrengthened by polymeric lignin covalently cross-linked tohemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thusa linear beta-(1-4)-D-glucan, while hemicelluloses include a variety ofcompounds, such as xylans, xyloglucans, arabinoxylans, and mannans incomplex branched structures with a spectrum of substituents. Althoughgenerally polymorphous, cellulose is found in plant tissue primarily asan insoluble crystalline matrix of parallel glucan chains.Hemicelluloses usually hydrogen bond to cellulose, as well as to otherhemicelluloses, which help stabilize the cell wall matrix.

Cellulose is generally found, for example, in the stems, leaves, hulls,husks, and cobs of plants or leaves, branches, and wood of trees. Thecellulosic material can be, but is not limited to, agricultural residue,herbaceous material (including energy crops), municipal solid waste,pulp and paper mill residue, waste paper, and wood (including forestryresidue) (see, for example, Wiselogel et al., 1995, in Handbook onBioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis,Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd,1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier etal., 1999, Recent Progress in Bioconversion of Lignocellulosics, inAdvances in Biochemical Engineering/Biotechnology, T. Scheper, managingeditor, Volume 65, pp. 23-40, Springer-Verlag, New York). It isunderstood herein that the cellulose may be in the form oflignocellulose, a plant cell wall material containing lignin, cellulose,and hemicellulose in a mixed matrix. In a preferred aspect, thecellulosic material is any biomass material. In another preferredaspect, the cellulosic material is lignocellulose, which comprisescellulose, hemicelluloses, and lignin.

In one aspect, the cellulosic material is agricultural residue. Inanother aspect, the cellulosic material is herbaceous material(including energy crops). In another aspect, the cellulosic material ismunicipal solid waste. In another aspect, the cellulosic material ispulp and paper mill residue. In another aspect, the cellulosic materialis waste paper. In another aspect, the cellulosic material is wood(including forestry residue).

In another aspect, the cellulosic material is arundo. In another aspect,the cellulosic material is bagasse. In another aspect, the cellulosicmaterial is bamboo. In another aspect, the cellulosic material is corncob. In another aspect, the cellulosic material is corn fiber. Inanother aspect, the cellulosic material is corn stover. In anotheraspect, the cellulosic material is miscanthus. In another aspect, thecellulosic material is orange peel. In another aspect, the cellulosicmaterial is rice straw. In another aspect, the cellulosic material isswitchgrass. In another aspect, the cellulosic material is wheat straw.

In another aspect, the cellulosic material is aspen. In another aspect,the cellulosic material is eucalyptus. In another aspect, the cellulosicmaterial is fir. In another aspect, the cellulosic material is pine. Inanother aspect, the cellulosic material is poplar. In another aspect,the cellulosic material is spruce. In another aspect, the cellulosicmaterial is willow.

In another aspect, the cellulosic material is algal cellulose. Inanother aspect, the cellulosic material is bacterial cellulose. Inanother aspect, the cellulosic material is cotton linter. In anotheraspect, the cellulosic material is filter paper. In another aspect, thecellulosic material is microcrystalline cellulose. In another aspect,the cellulosic material is phosphoric-acid treated cellulose.

In another aspect, the cellulosic material is an aquatic biomass. Asused herein the term “aquatic biomass” means biomass produced in anaquatic environment by a photosynthesis process. The aquatic biomass canbe algae, emergent plants, floating-leaf plants, or submerged plants.

The cellulosic material may be used as is or may be subjected topretreatment, using conventional methods known in the art, as describedherein. In a preferred aspect, the cellulosic material is pretreated.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which begins with a start codon such as ATG, GTG, or TTGand ends with a stop codon such as TAA, TAG, or TGA. The coding sequencemay be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other. Such control sequences include, but are notlimited to, a leader, polyadenylation sequence, propeptide sequence,promoter, signal peptide sequence, and transcription terminator. At aminimum, the control sequences include a promoter, and transcriptionaland translational stop signals. The control sequences may be providedwith linkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe polynucleotide encoding a polypeptide.

Endoglucanase: The term “endoglucanase” means anendo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) thatcatalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose,cellulose derivatives (such as carboxymethyl cellulose and hydroxyethylcellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such ascereal beta-D-glucans or xyloglucans, and other plant materialcontaining cellulosic components. Endoglucanase activity can bedetermined by measuring reduction in substrate viscosity or increase inreducing ends determined by a reducing sugar assay (Zhang et al., 2006,Biotechnology Advances 24: 452-481). For purposes of the presentinvention, endoglucanase activity is determined using carboxymethylcellulose (CMC) as substrate according to the procedure of Ghose, 1987,Pure and Appl. Chem. 59: 257-268, at pH 5, 40° C.

Expression: The term “expression” includes any step involved in theproduction of a polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to control sequences that provide forits expression.

Family 61 glycoside hydrolase: The term “Family 61 glycoside hydrolase”or “Family GH61” or “GH61” means a polypeptide falling into theglycoside hydrolase Family 61 according to Henrissat B., 1991, Aclassification of glycosyl hydrolases based on amino-acid sequencesimilarities, Biochem. J. 280: 309-316, and Henrissat B., and BairochA., 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696. The enzymes in this family wereoriginally classified as a glycoside hydrolase family based onmeasurement of very weak endo-1,4-beta-D-glucanase activity in onefamily member. The structure and mode of action of these enzymes arenon-canonical and they cannot be considered as bona fide glycosidases.However, they are kept in the CAZy classification on the basis of theircapacity to enhance the breakdown of lignocellulose when used inconjunction with a cellulase or a mixture of cellulases.

Feruloyl esterase: The term “feruloyl esterase” means a4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) thatcatalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl)groups from esterified sugar, which is usually arabinose in naturalbiomass substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate).Feruloyl esterase is also known as ferulic acid esterase,hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA,cinnAE, FAE-I, or FAE-II. For purposes of the present invention,feruloyl esterase activity is determined using 0.5 mMp-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. Oneunit of feruloyl esterase equals the amount of enzyme capable ofreleasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

Fragment: The term “fragment” means a polypeptide or a catalytic orcellulose binding domain having one or more (e.g., several) amino acidsabsent from the amino and/or carboxyl terminus of a mature polypeptideor domain; wherein the fragment has cellobiohydrolase or cellulosebinding activity. In one aspect, a fragment contains at least 375 aminoacid residues, e.g., at least 395 amino acid residues or at least 415amino acid residues.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolyticenzyme” or “hemicellulase” means one or more (e.g., several) enzymesthat hydrolyze a hemicellulosic material. See, for example, Shallom andShoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology6(3): 219-228). Hemicellulases are key components in the degradation ofplant biomass. Examples of hemicellulases include, but are not limitedto, an acetylmannan esterase, an acetylxylan esterase, an arabinanase,an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, agalactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, amannosidase, a xylanase, and a xylosidase. The substrates of theseenzymes, the hemicelluloses, are a heterogeneous group of branched andlinear polysaccharides that are bound via hydrogen bonds to thecellulose microfibrils in the plant cell wall, crosslinking them into arobust network. Hemicelluloses are also covalently attached to lignin,forming together with cellulose a highly complex structure. The variablestructure and organization of hemicelluloses require the concertedaction of many enzymes for its complete degradation. The catalyticmodules of hemicellulases are either glycoside hydrolases (GHs) thathydrolyze glycosidic bonds, or carbohydrate esterases (CEs), whichhydrolyze ester linkages of acetate or ferulic acid side groups. Thesecatalytic modules, based on homology of their primary sequence, can beassigned into GH and CE families. Some families, with an overall similarfold, can be further grouped into clans, marked alphabetically (e.g.,GH-A). A most informative and updated classification of these and othercarbohydrate active enzymes is available in the Carbohydrate-ActiveEnzymes (CAZy) database. Hemicellulolytic enzyme activities can bemeasured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59:1739-1752, at a suitable temperature, e.g., 50° C., 55° C., or 60° C.,and pH, e.g., 5.0 or 5.5.

High stringency conditions: The term “high stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 50% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at65° C.

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, or the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated: The term “isolated” means a substance in a form or environmentthat does not occur in nature. Non-limiting examples of isolatedsubstances include (1) any non-naturally occurring substance, (2) anysubstance including, but not limited to, any enzyme, variant, nucleicacid, protein, peptide or cofactor, that is at least partially removedfrom one or more or all of the naturally occurring constituents withwhich it is associated in nature; (3) any substance modified by the handof man relative to that substance found in nature; or (4) any substancemodified by increasing the amount of the substance relative to othercomponents with which it is naturally associated (e.g., multiple copiesof a gene encoding the substance; use of a stronger promoter than thepromoter naturally associated with the gene encoding the substance). Thepolypeptide of the present invention may be used in industrialapplications in the form of a fermentation broth product, that is, thepolypeptide of the present invention is a component of a fermentationbroth used as a product in industrial applications (e.g., ethanolproduction). The fermentation broth product will in addition to thepolypeptide of the present invention comprise additional ingredientsused in the fermentation process, such as, for example, cells(including, the host cells containing the gene encoding the polypeptideof the present invention which are used to produce the polypeptide ofinterest), cell debris, biomass, fermentation media and/or fermentationproducts. The fermentation broth may optionally be subjected to one ormore purification (including filtration) steps to remove or reduce onemore components of a fermentation process. Accordingly, an isolatedsubstance may be present in such a fermentation broth product.

Low stringency conditions: The term “low stringency conditions” meansfor probes of at least 100 nucleotides in length, prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml shearedand denatured salmon sperm DNA, and 25% formamide, following standardSouthern blotting procedures for 12 to 24 hours. The carrier material isfinally washed three times each for 15 minutes using 2×SSC, 0.2% SDS at50° C.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 20 to 456 of SEQ ID NO: 2 based on theSignalP program (Nielsen et al., 1997, Protein Engineering 10: 1-6) thatpredicts amino acids 1 to 19 of SEQ ID NO: 2 are a signal peptide. It isknown in the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving cellobiohydrolase activity. In one aspect, the mature polypeptidecoding sequence is nucleotides 58 to 1786 of SEQ ID NO: 1 or the cDNAsequence thereof based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 57 of SEQ ID NO: 1 encode a signalpeptide.

Medium stringency conditions: The term “medium stringency conditions”means for probes of at least 100 nucleotides in length, prehybridizationand hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/mlsheared and denatured salmon sperm DNA, and 35% formamide, followingstandard Southern blotting procedures for 12 to 24 hours. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 55° C.

Medium-high stringency conditions: The term “medium-high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 60° C.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic, which comprises one or more controlsequences.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs expression of the coding sequence.

Polypeptide having cellulolytic enhancing activity: The term“polypeptide having cellulolytic enhancing activity” means a GH61polypeptide that catalyzes the enhancement of the hydrolysis of acellulosic material by enzyme having cellulolytic activity. For purposesof the present invention, cellulolytic enhancing activity is determinedby measuring the increase in reducing sugars or the increase of thetotal of cellobiose and glucose from the hydrolysis of a cellulosicmaterial by cellulolytic enzyme under the following conditions: 1-50 mgof total protein/g of cellulose in PCS, wherein total protein iscomprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/wprotein of a GH61 polypeptide having cellulolytic enhancing activity for1-7 days at a suitable temperature, e.g., 50° C., 55° C., or 60° C., andpH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal totalprotein loading without cellulolytic enhancing activity (1-50 mg ofcellulolytic protein/g of cellulose in PCS). In a preferred aspect, amixture of CELLUCLAST@ 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in thepresence of 2-3% of total protein weight Aspergillus otyzaebeta-glucosidase (recombinantly produced in Aspergillus otyzae accordingto WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatusbeta-glucosidase (recombinantly produced in Aspergillus olyzae asdescribed in WO 2002/095014) of cellulase protein loading is used as thesource of the cellulolytic activity.

The GH61 polypeptides having cellulolytic enhancing activity enhance thehydrolysis of a cellulosic material catalyzed by enzyme havingcellulolytic activity by reducing the amount of cellulolytic enzymerequired to reach the same degree of hydrolysis preferably at least1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

Pretreated corn stover: The term “PCS” or “Pretreated Corn Stover” meansa cellulosic material derived from corn stover by treatment with heatand dilute sulfuric acid, alkaline pretreatment, or neutralpretreatment.

Sequence identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the sequence identity between twoamino acid sequences is determined using the Needleman-Wunsch algorithm(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implementedin the Needle program of the EMBOSS package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 5.0.0 or later. The parameters used aregap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62(EMBOSS version of BLOSUM62) substitution matrix. The output of Needlelabeled “longest identity” (obtained using the nobrief option) is usedas the percent identity and is calculated as follows:(Identical Residues×100)/(Length of Alignment Total Number of Gaps inAlignment)

For purposes of the present invention, the sequence identity between twodeoxyribonucleotide sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, supra) as implemented in theNeedle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, supra), preferablyversion 5.0.0 or later. The parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the nobrief option) is used as the percentidentity and is calculated as follows:(Identical Deoxyribonucleotides×100)/(Length of Alignment Total Numberof Gaps in Alignment)

Subsequence: The term “subsequence” means a polynucleotide having one ormore (e.g., several) nucleotides absent from the 5′ and/or 3′ end of amature polypeptide coding sequence; wherein the subsequence encodes afragment having cellobiohydrolase activity. In one aspect, a subsequencecontains at least 1125 nucleotides, e.g., at least 1185 nucleotides orat least 1245 nucleotides; or the cDNA sequence thereof.

Variant: The term “variant” means a polypeptide having cellobiohydrolaseactivity comprising an alteration, i.e., a substitution, insertion,and/or deletion, at one or more (e.g., several) positions. Asubstitution means replacement of the amino acid occupying a positionwith a different amino acid; a deletion means removal of the amino acidoccupying a position; and an insertion means adding an amino acidadjacent to and immediately following the amino acid occupying aposition.

Very high stringency conditions: The term “very high stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 70° C.

Very low stringency conditions: The term “very low stringencyconditions” means for probes of at least 100 nucleotides in length,prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide,following standard Southern blotting procedures for 12 to 24 hours. Thecarrier material is finally washed three times each for 15 minutes using2×SSC, 0.2% SDS at 45° C.

Xylan-containing material: The term “xylan-containing material” meansany material comprising a plant cell wall polysaccharide containing abackbone of beta-(1-4)-linked xylose residues. Xylans of terrestrialplants are heteropolymers possessing a beta-(1-4)-D-xylopyranosebackbone, which is branched by short carbohydrate chains. They compriseD-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or variousoligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose,and D-glucose. Xylan-type polysaccharides can be divided into homoxylansand heteroxylans, which include glucuronoxylans,(arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, andcomplex heteroxylans. See, for example, Ebringerova et al., 2005, Adv.Polym. Sci. 186: 1-67.

In the methods of the present invention, any material containing xylanmay be used. In a preferred aspect, the xylan-containing material islignocellulose.

Xylan degrading activity or xylanolytic activity: The term “xylandegrading activity” or “xylanolytic activity” means a biologicalactivity that hydrolyzes xylan-containing material. The two basicapproaches for measuring xylanolytic activity include: (1) measuring thetotal xylanolytic activity, and (2) measuring the individual xylanolyticactivities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases,alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, andalpha-glucuronyl esterases). Recent progress in assays of xylanolyticenzymes was summarized in several publications including Biely andPuchard, 2006, Recent progress in the assays of xylanolytic enzymes,Journal of the Science of Food and Agriculture 86(11): 1636-1647;Spanikova and Biely, 2006, Glucuronoyl esterase—Novel carbohydrateesterase produced by Schizophyllum commune, FEBS Letters 580(19):4597-4601; Herrmann et al., 1997, The beta-D-xylosidase of Trichodermareesei is a multifunctional beta-D-xylan xylohydrolase, BiochemicalJournal 321: 375-381.

Total xylan degrading activity can be measured by determining thereducing sugars formed from various types of xylan, including, forexample, oat spelt, beechwood, and larchwood xylans, or by photometricdetermination of dyed xylan fragments released from various covalentlydyed xylans. The most common total xylanolytic activity assay is basedon production of reducing sugars from polymeric 4-O-methylglucuronoxylan as described in Bailey et al., 1992, Interlaboratorytesting of methods for assay of xylanase activity, Journal ofBiotechnology 23(3): 257-270. Xylanase activity can also be determinedwith 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON® X-100(4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mMsodium phosphate buffer pH 6 at 37° C. One unit of xylanase activity isdefined as 1.0 μmole of azurine produced per minute at 37° C., pH 6 from0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6buffer.

For purposes of the present invention, xylan degrading activity isdetermined by measuring the increase in hydrolysis of birchwood xylan(Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degradingenzyme(s) under the following typical conditions: 1 ml reactions, 5mg/ml substrate (total solids), 5 mg of xylanolytic protein/g ofsubstrate, 50 mM sodium acetate pH 5, 50° C., 24 hours, sugar analysisusing p-hydroxybenzoic acid hydrazide (PHBAH) assay as described byLever, 1972, A new reaction for colorimetric determination ofcarbohydrates, Anal. Biochem 47: 273-279.

Xylanase: The term “xylanase” means a 1,4-beta-D-xylan-xylohydrolase(E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidiclinkages in xylans. For purposes of the present invention, xylanaseactivity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01%TRITON® X-100 and 200 mM sodium phosphate buffer pH 6 at 37° C. One unitof xylanase activity is defined as 1.0 μmole of azurine produced perminute at 37° C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides HavingCellobiohydrolase Activity

In an embodiment, the present invention relates to isolated polypeptideshaving a sequence identity to the mature polypeptide of SEQ ID NO: 2 ofat least 85%, e.g., at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%, which have cellobiohydrolase activity. In one aspect, thepolypeptides differ by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, or 10, from the mature polypeptide of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity. Inanother aspect, the polypeptide comprises or consists of the maturepolypeptide of SEQ ID NO: 2. In another aspect, the polypeptidecomprises or consists of amino acids 20 to 456 of SEQ ID NO: 2.

In another embodiment, the present invention relates to isolatedpolypeptides having cellobiohydrolase activity that are encoded bypolynucleotides that hybridize under very low stringency conditions, lowstringency conditions, medium stringency conditions, medium-highstringency conditions, high stringency conditions, or very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2d edition, Cold Spring Harbor, New York).

The polynucleotide of SEQ ID NO: 1 or a subsequence thereof, as well asthe polypeptide of SEQ ID NO: 2 or a fragment thereof, may be used todesign nucleic acid probes to identify and clone DNA encodingpolypeptides having cellobiohydrolase activity from strains of differentgenera or species according to methods well known in the art. Inparticular, such probes can be used for hybridization with the genomicDNA or cDNA of a cell of interest, following standard Southern blottingprocedures, in order to identify and isolate the corresponding genetherein. Such probes can be considerably shorter than the entiresequence, but should be at least 15, e.g., at least 25, at least 35, orat least 70 nucleotides in length. Preferably, the nucleic acid probe isat least 100 nucleotides in length, e.g., at least 200 nucleotides, atleast 300 nucleotides, at least 400 nucleotides, at least 500nucleotides, at least 600 nucleotides, at least 700 nucleotides, atleast 800 nucleotides, or at least 900 nucleotides in length. Both DNAand RNA probes can be used. The probes are typically labeled fordetecting the corresponding gene (for example, with ³²P, ³H, ³⁵S,biotin, or avidin). Such probes are encompassed by the presentinvention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having cellobiohydrolase activity. Genomic orother DNA from such other strains may be separated by agarose orpolyacrylamide gel electrophoresis, or other separation techniques. DNAfrom the libraries or the separated DNA may be transferred to andimmobilized on nitrocellulose or other suitable carrier material. Inorder to identify a clone or DNA that hybridzes with SEQ ID NO: 1 or asubsequence thereof, the carrier material is used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labeled nucleic acid probe correspondingto (i) SEQ ID NO: 1; (ii) the mature polypeptide coding sequence of SEQID NO: 1; (iii) the cDNA sequence thereof; (iv) the full-lengthcomplement thereof; or (v) a subsequence thereof; under very low to veryhigh stringency conditions. Molecules to which the nucleic acid probehybridizes under these conditions can be detected using, for example,X-ray film or any other detection means known in the art.

In one aspect, the nucleic acid probe is nucleotides 58 to 1786 of SEQID NO: 1 or the cDNA thereof. In another aspect, the nucleic acid probeis a polynucleotide that encodes the polypeptide of SEQ ID NO: 2 or themature polypeptide thereof; or a fragment thereof. In another aspect,the nucleic acid probe is SEQ ID NO: 1 or the cDNA sequence thereof. Inanother aspect, the nucleic acid probe is the polynucleotide containedin plasmid pAJ227 which is contained in E. coli NRRL B-50474, whereinthe polynucleotide encodes a polypeptide having cellobiohydrolaseactivity. In another aspect, the nucleic acid probe is the maturepolypeptide coding region contained in plasmid pAJ227 which is containedin E. coli NRRL B-50474.

In another embodiment, the present invention relates to isolatedpolypeptides having cellobiohydrolase activity encoded bypolynucleotides having a sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof of at least85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to variants of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more (e.g., several) positions. In anembodiment, the number of amino acid substitutions, deletions and/orinsertions introduced into the mature polypeptide of SEQ ID NO: 2 is upto 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changesmay be of a minor nature, that is conservative amino acid substitutionsor insertions that do not significantly affect the folding and/oractivity of the protein; small deletions, typically of 1-30 amino acids;small amino- or carboxyl-terminal extensions, such as an amino-terminalmethionine residue; a small linker peptide of up to 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tract, an antigenic epitopeor a binding domain.

Examples of conservative substitutions are within the groups of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. Commonsubstitutions are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr,Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile,Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a polypeptide can be identified according toprocedures known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244:1081-1085). In the latter technique, single alanine mutations areintroduced at every residue in the molecule, and the resultant mutantmolecules are tested for cellobiohydrolase activity to identify aminoacid residues that are critical to the activity of the molecule. Seealso, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity ofessential amino acids can also be inferred from an alignment with arelated polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The polypeptide may be a hybrid polypeptide in which a region of onepolypeptide is fused at the N-terminus or the C-terminus of a region ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusionpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fusion polypeptide is under control of thesame promoter(s) and terminator. Fusion polypeptides may also beconstructed using intein technology in which fusion polypeptides arecreated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

Sources of Polypeptides Having Cellobiohydrolase Activity

A polypeptide having cellobiohydrolase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by apolynucleotide is produced by the source or by a strain in which thepolynucleotide from the source has been inserted. In one aspect, thepolypeptide obtained from a given source is secreted extracellularly.

The polypeptide may be a bacterial polypeptide. For example, thepolypeptide may be a Gram positive bacterial polypeptide such as aBacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, orStreptomyces polypeptide having cellobiohydrolase activity, or a Gramnegative bacterial polypeptide such as a Campylobacter, E. coli,Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria,Pseudomonas, Salmonella, or Ureaplasma polypeptide.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide.

The polypeptide may also be a fungal polypeptide. For example, thepolypeptide may be a yeast polypeptide such as a Candida, Kluyveromyces,Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; ora filamentous fungal polypeptide such as an Acremonium, Agaricus,Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis,Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis,Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia,Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex,Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudo plectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thiela via, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide.

In another aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasii, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus,Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium mops, Chrysosporiumkeratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium,Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporiumtropicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechlysosporium, Talaromyces byssochlamydoides, Talaromyces emersonii,Talaromyces stipitatus, Thielavia achromatica, Thielavia albomyces,Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti,Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielaviasetosa, Thielavia spededonium, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide.

In another aspect, the polypeptide is a Talaromyces byssochlamydoidespolypeptide, e.g., a polypeptide obtained from Talaromycesbyssochlamydoides CBS 413.71.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen undZellkulturen GmbH (DSMZ), Centraalbureau Voor Schimmelcultures (CBS),and Agricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Catalytic Domains

In one embodiment, the present invention also relates to catalyticdomains having a sequence identity to amino acids 98 to 456 of SEQ IDNO: 2 of at least 90%, e.g., at least 91%, at least 92%, at least 93%,at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%. In another embodiment, the catalytic domainscomprise amino acid sequences that differ by up to 10 amino acids, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 98 to 456 of SEQ IDNO: 2.

In a preferred embodiment, the catalytic domain preferably comprises orconsists of amino acids 98 to 456 of SEQ ID NO: 2 or an allelic variantthereof; or is a fragment thereof having cellobiohydrolase activity.

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) with(i) nucleotides 397 to 1786 of SEQ ID NO: 1, (ii) the cDNA sequencethereof, or (iii) the full-length complement of (i) or (ii) (Sambrook etal., 1989, supra).

In another embodiment, the present invention also relates to catalyticdomains encoded by polynucleotides having a sequence identity tonucleotides 397 to 1786 of SEQ ID NO: 1 or the cDNA thereof of at least90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In a preferred embodiment, the polynucleotide encoding the catalyticdomain preferably comprises or consists of nucleotides 397 to 1786 ofSEQ ID NO: 1 or is the sequence contained in plasmid pAJ227 which iscontained in E. coli NRRL B-50474.

In another embodiment, the present invention also relates to catalyticdomain variants of amino acids 98 to 456 of SEQ ID NO: 2 comprising asubstitution, deletion, and/or insertion at one or more (e.g., several)positions. In another embodiment, the number of amino acidsubstitutions, deletions and/or insertions introduced into the sequenceof amino acids 98 to 456 of SEQ ID NO: 2 is 10, e.g., 1, 2, 3, 4, 5, 6,8, 9, or 10.

Binding Domains

In one embodiment, the present invention also relates to cellulosebinding domains having a sequence identity to amino acids 20 to 56 ofSEQ ID NO: 2 of at least 90%, e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100%. In another embodiment, the cellulose bindingdomains comprise amino acid sequences that differ by up to 10 aminoacids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, from amino acids 20 to 56of SEQ ID NO: 2.

In a preferred embodiment, the cellulose binding domain preferablycomprises or consists of amino acids 20 to 56 of SEQ ID NO: 2 or anallelic variant thereof; or is a fragment thereof having cellulosebinding activity.

In another embodiment, the present invention also relates to cellulosebinding domains encoded by polynucleotides that hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions (as defined above) with(i) the nucleotides 58 to 273 of SEQ ID NO: 1, (ii) the cDNA sequence ofnucleotides 58 to 273 of SEQ ID NO: 1, or (iii) the full-lengthcomplement of (i) or (ii) (Sambrook et al., 1989, supra).

In another embodiment, the present invention also relates to cellulosebinding domains encoded by polynucleotides having a sequence identity tonucleotides 58 to 273 of SEQ ID NO: 1 or the cDNA thereof of at least90%, e.g., at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In a preferred embodiment, the polynucleotide encoding the cellulosebinding domain comprises or consists of nucleotides 58 to 273 of SEQ IDNO: 1 or is the sequence contained in plasmid pAJ227 which is containedin E. coli NRRL B-50474.

In another embodiment, the present invention also relates to cellulosebinding domain variants of amino acids 20 to 56 of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or more(e.g., several) positions. In another embodiment, the number of aminoacid substitutions, deletions and/or insertions introduced into thesequence of amino acids 20 to 56 of SEQ ID NO: 2 is 10, e.g., 1, 2, 3,4, 5, 6, 8, 9, or 10.

A catalytic domain operably linked to the binding domain may be from ahydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase,e.g., an aminopeptidase, amylase, carbohydrase, carboxypeptidase,catalase, cellobiohydrolase, cellulase, chitinase, cutinase,cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase,esterase, alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, xylanase, or beta-xylosidase. The polynucleotideencoding the catalytic domain may be obtained from any prokaryotic,eukaryotic, or other source.

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide, a catalytic domain, or a cellulose binding domain of thepresent invention, as described herein.

The techniques used to isolate or clone a polynucleotide are known inthe art and include isolation from genomic DNA or cDNA, or a combinationthereof. The cloning of the polynucleotides from genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligation activated transcription (LAT) andpolynucleotide-based amplification (NASBA) may be used. Thepolynucleotides may be cloned from a strain of Talaromyces, or a relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the polynucleotide.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for synthesizing polypeptides substantiallysimilar to the polypeptide. The term “substantially similar” to thepolypeptide refers to non-naturally occurring forms of the polypeptide.These polypeptides may differ in some engineered way from thepolypeptide isolated from its native source, e.g., variants that differin specific activity, thermostability, pH optimum, or the like. Thevariants may be constructed on the basis of the polynucleotide presentedas the mature polypeptide coding sequence of SEQ ID NO: 1 or the cDNAsequence thereof and/or by introduction of nucleotide substitutions thatdo not result in a change in the amino acid sequence of the polypeptide,but which correspond to the codon usage of the host organism intendedfor production of the enzyme, or by introduction of nucleotidesubstitutions that may give rise to a different amino acid sequence. Fora general description of nucleotide substitution, see, e.g., Ford etal., 1991, Protein Expression and Purification 2: 95-107.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the expression of the coding sequence in asuitable host cell under conditions compatible with the controlsequences.

The polynucleotide may be manipulated in a variety of ways to providefor expression of the polypeptide. Manipulation of the polynucleotideprior to its insertion into a vector may be desirable or necessarydepending on the expression vector. The techniques for modifyingpolynucleotides utilizing recombinant DNA methods are well known in theart.

The control sequence may be a promoter, a polynucleotide that isrecognized by a host cell for expression of a polynucleotide encoding apolypeptide of the present invention. The promoter containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell including mutant, truncated,and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryllIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dana (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIll, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor, as well as the NA2-tpi promoter (a modified promoterfrom an Aspergillus neutral alpha-amylase gene in which the untranslatedleader has been replaced by an untranslated leader from an Aspergillustriose phosphate isomerase gene; non-limiting examples include modifiedpromoters from an Aspergillus niger neutral alpha-amylase gene in whichthe untranslated leader has been replaced by an untranslated leader froman Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerasegene); and mutant, truncated, and hybrid promoters thereof. Otherpromoters are described in U.S. Pat. No. 6,011,147.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a transcription terminator, which isrecognized by a host cell to terminate transcription. The terminator isoperably linked to the 3′-terminus of the polynucleotide encoding thepolypeptide. Any terminator that is functional in the host cell may beused in the present invention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans acetamidase, Aspergillusnidulans anthranilate synthase, Aspergillus niger glucoamylase,Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase,Fusarium oxysporum trypsin-like protease, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I,Trichoderma reesei xylanase II, Trichoderma reesei xylanase III,Trichoderma reesei beta-xylosidase, and Trichoderma reesei translationelongation factor.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis crylIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus nidulans anthranilatesynthase, Aspergillus niger glucoamylase, Aspergillus nigeralpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusariumoxysporum trypsin-like protease.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. A foreign signal peptide coding sequence may be required wherethe coding sequence does not naturally contain a signal peptide codingsequence. Alternatively, a foreign signal peptide coding sequence maysimply replace the natural signal peptide coding sequence in order toenhance secretion of the polypeptide. However, any signal peptide codingsequence that directs the expressed polypeptide into the secretorypathway of a host cell may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, thepropeptide sequence is positioned next to the N-terminus of apolypeptide and the signal peptide sequence is positioned next to theN-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that regulateexpression of the polypeptide relative to the growth of the host cell.Examples of regulatory sequences are those that cause expression of thegene to be turned on or off in response to a chemical or physicalstimulus, including the presence of a regulatory compound. Regulatorysequences in prokaryotic systems include the lac, tac, and trp operatorsystems. In yeast, the ADH2 system or GAL1 system may be used. Infilamentous fungi, the Aspergillus niger glucoamylase promoter,Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzaeglucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter,and Trichoderma reesei cellobiohydrolase II promoter may be used. Otherexamples of regulatory sequences are those that allow for geneamplification. In eukaryotic systems, these regulatory sequences includethe dihydrofolate reductase gene that is amplified in the presence ofmethotrexate, and the metallothionein genes that are amplified withheavy metals. In these cases, the polynucleotide encoding thepolypeptide would be operably linked to the regulatory sequence.Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more convenient restrictionsites to allow for insertion or substitution of the polynucleotideencoding the polypeptide at such sites. Alternatively, thepolynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the polynucleotide into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more selectable markers thatpermit easy selection of transformed, transfected, transduced, or thelike cells. A selectable marker is a gene the product of which providesfor biocide or viral resistance, resistance to heavy metals, prototrophyto auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HI53, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, adeA (phosphoribosylaminoimidazole-succinocarboxam ide synthase), adeB(phosphoribosyl-aminoimidazole synthase), amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene. Preferred for use in a Trichodermacell are adeA, adeB, amdS, hph, and pyrG genes.

The selectable marker may be a dual selectable marker system asdescribed in WO 2010/039889. In one aspect, the dual selectable markeris a hph-tk dual selectable marker system.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAN/1111permitting replication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or morecontrol sequences that direct the production of a polypeptide of thepresent invention. A construct or vector comprising a polynucleotide isintroduced into a host cell so that the construct or vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram positive or Gram negativebacterium. Gram positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacilluslicheniformis, Bacillus megaterium, Bacillus pumilus, Bacillusstearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may be effected byprotoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen.Genet. 168: 111-115), competent cell transformation (see, e.g., Youngand Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), orconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may be effectedby protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol.166: 557-580) or electroporation (see, e.g., Dower et al., 1988, NucleicAcids Res. 16: 6127-6145). The introduction of DNA into a Streptomycescell may be effected by protoplast transformation, electroporation (see,e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405),conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or transduction (see, e.g., Burke et al., 2001, Proc. Natl.Acad. Sci. USA 98: 6289-6294). The introduction of DNA into aPseudomonas cell may be effected by electroporation (see, e.g., Choi etal., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g.,Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). Theintroduction of DNA into a Streptococcus cell may be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect lmmun. 32:1295-1297), protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota as wellas the Oomycota and all mitosporic fungi (as defined by Hawksworth etal., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,1995, CAB International, University Press, Cambridge, UK).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi lmperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, Passmore, and Davenport,editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thiela via, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phlebia radiata, Pleurotus etyngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermahatzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422.Suitable methods for transforming Fusarium species are described byMalardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may betransformed using the procedures described by Becker and Guarente, InAbelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics andMolecular Biology, Methods in Enzymology, Volume 194, pp 182-187,Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol. 153:163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In one aspect, the cell is a Talaromyces cell. In another aspect, thecell is a Talaromyces byssochlamydoides cell. In another aspect, thecell is Talaromyces byssochlamydoides CBS 413.71.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The cell is cultivated in a nutrient medium suitable for production ofthe polypeptide using methods known in the art. For example, the cellmay be cultivated by shake flask cultivation, or small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentors in asuitable medium and under conditions allowing the polypeptide to beexpressed and/or isolated. The cultivation takes place in a suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media are availablefrom commercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted, it can be recovered from cell lysates.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods include, but arenot limited to, use of specific antibodies, formation of an enzymeproduct, or disappearance of an enzyme substrate. For example, an enzymeassay may be used to determine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, collection,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, Janson andRyden, editors, VCH Publishers, New York, 1989) to obtain substantiallypure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing the polypeptide is used asa source of the polypeptide.

Plants

The present invention also relates to isolated plants, e.g., atransgenic plant, plant part, or plant cell, comprising a polynucleotideof the present invention so as to express and produce a polypeptide ordomain in recoverable quantities. The polypeptide or domain may berecovered from the plant or plant part. Alternatively, the plant orplant part containing the polypeptide or domain may be used as such forimproving the quality of a food or feed, e.g., improving nutritionalvalue, palatability, and rheological properties, or to destroy anantinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seed coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing the polypeptide or domainmay be constructed in accordance with methods known in the art. Inshort, the plant or plant cell is constructed by incorporating one ormore expression constructs encoding the polypeptide or domain into theplant host genome or chloroplast genome and propagating the resultingmodified plant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide or domain operablylinked with appropriate regulatory sequences required for expression ofthe polynucleotide in the plant or plant part of choice. Furthermore,the expression construct may comprise a selectable marker useful foridentifying plant cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide or domainis desired to be expressed. For instance, the expression of the geneencoding a polypeptide or domain may be constitutive or inducible, ormay be developmental, stage or tissue specific, and the gene product maybe targeted to a specific tissue or plant part such as seeds or leaves.Regulatory sequences are, for example, described by Tague et al., 1988,Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, or therice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be induced by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide or domain in the plant. For instance, thepromoter enhancer element may be an intron that is placed between thepromoter and the polynucleotide encoding a polypeptide or domain. Forinstance, Xu et al., 1993, supra, disclose the use of the first intronof the rice actin 1 gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Agrobacterium tumefaciens-mediated gene transfer is a method forgenerating transgenic dicots (for a review, see Hooykas andSchilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transformingmonocots, although other transformation methods may be used for theseplants. A method for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 5:158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternativemethod for transformation of monocots is based on protoplasttransformation as described by Omirulleh et al., 1993, Plant Mol. Biol.21: 415-428. Additional transformation methods include those describedin U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are hereinincorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct of the present invention, transgenic plants may be made bycrossing a plant having the construct to a second plant lacking theconstruct. For example, a construct encoding a polypeptide or domain canbe introduced into a particular plant variety by crossing, without theneed for ever directly transforming a plant of that given variety.Therefore, the present invention encompasses not only a plant directlyregenerated from cells which have been transformed in accordance withthe present invention, but also the progeny of such plants. As usedherein, progeny may refer to the offspring of any generation of a parentplant prepared in accordance with the present invention. Such progenymay include a DNA construct prepared in accordance with the presentinvention. Crossing results in the introduction of a transgene into aplant line by cross pollinating a starting line with a donor plant line.Non-limiting examples of such steps are described in U.S. Pat. No.7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideor domain of the present invention comprising (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encodingthe polypeptide or domain under conditions conducive for production ofthe polypeptide or domain; and (b) recovering the polypeptide or domain.

Removal or Reduction of Cellobiohydrolase Activity

The present invention also relates to methods of producing a mutant of aparent cell, which comprises disrupting or deleting a polynucleotide, ora portion thereof, encoding a polypeptide of the present invention,which results in the mutant cell producing less of the polypeptide thanthe parent cell when cultivated under the same conditions.

The mutant cell may be constructed by reducing or eliminating expressionof the polynucleotide using methods well known in the art, for example,insertions, disruptions, replacements, or deletions. In a preferredaspect, the polynucleotide is inactivated. The polynucleotide to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required forexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thepolynucleotide. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the polynucleotide may be performed bysubjecting the parent cell to mutagenesis and selecting for mutant cellsin which expression of the polynucleotide has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the polynucleotide may be accomplishedby insertion, substitution, or deletion of one or more nucleotides inthe gene or a regulatory element required for transcription ortranslation thereof. For example, nucleotides may be inserted or removedso as to result in the introduction of a stop codon, the removal of thestart codon, or a change in the open reading frame. Such modification orinactivation may be accomplished by site-directed mutagenesis or PCRgenerated mutagenesis in accordance with methods known in the art.Although, in principle, the modification may be performed in vivo, i.e.,directly on the cell expressing the polynucleotide to be modified, it ispreferred that the modification be performed in vitro as exemplifiedbelow.

An example of a convenient way to eliminate or reduce expression of apolynucleotide is based on techniques of gene replacement, genedeletion, or gene disruption. For example, in the gene disruptionmethod, a nucleic acid sequence corresponding to the endogenouspolynucleotide is mutagenized in vitro to produce a defective nucleicacid sequence that is then transformed into the parent cell to produce adefective gene. By homologous recombination, the defective nucleic acidsequence replaces the endogenous polynucleotide. It may be desirablethat the defective polynucleotide also encodes a marker that may be usedfor selection of transformants in which the polynucleotide has beenmodified or destroyed. In an aspect, the polynucleotide is disruptedwith a selectable marker such as those described herein.

The present invention also relates to methods of inhibiting theexpression of a polypeptide having cellobiohydrolase activity in a cell,comprising administering to the cell or expressing in the cell adouble-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises asubsequence of a polynucleotide of the present invention. In a preferredaspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 ormore duplex nucleotides in length.

The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA(miRNA). In a preferred aspect, the dsRNA is small interfering RNA forinhibiting transcription. In another preferred aspect, the dsRNA ismicro RNA for inhibiting translation.

The present invention also relates to such double-stranded RNA (dsRNA)molecules, comprising a portion of the mature polypeptide codingsequence of SEQ ID NO: 1 for inhibiting expression of the polypeptide ina cell. While the present invention is not limited by any particularmechanism of action, the dsRNA can enter a cell and cause thedegradation of a single-stranded RNA (ssRNA) of similar or identicalsequences, including endogenous mRNAs. When a cell is exposed to dsRNA,mRNA from the homologous gene is selectively degraded by a processcalled RNA interference (RNAi).

The dsRNAs of the present invention can be used in gene-silencing. Inone aspect, the invention provides methods to selectively degrade RNAusing a dsRNAi of the present invention. The process may be practiced invitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can beused to generate a loss-of-function mutation in a cell, an organ or ananimal. Methods for making and using dsRNA molecules to selectivelydegrade RNA are well known in the art; see, for example, U.S. Pat. Nos.6,489,127; 6,506,559; 6,511,824; and 6,515,109.

The present invention further relates to a mutant cell of a parent cellthat comprises a disruption or deletion of a polynucleotide encoding thepolypeptide or a control sequence thereof or a silenced gene encodingthe polypeptide, which results in the mutant cell producing less of thepolypeptide or no polypeptide compared to the parent cell.

The polypeptide-deficient mutant cells are particularly useful as hostcells for expression of native and heterologous polypeptides. Therefore,the present invention further relates to methods of producing a nativeor heterologous polypeptide, comprising (a) cultivating the mutant cellunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide. The term “heterologous polypeptides” meanspolypeptides that are not native to the host cell, e.g., a variant of anative protein. The host cell may comprise more than one copy of apolynucleotide encoding the native or heterologous polypeptide.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallycellobiohydrolase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The cellobiohydrolase-deficient cells may also be usedto express heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like. The term “eukaryoticpolypeptides” includes not only native polypeptides, but also thosepolypeptides, e.g., enzymes, which have been modified by amino acidsubstitutions, deletions or additions, or other such modifications toenhance activity, thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from cellobiohydrolase activity that is produced by amethod of the present invention.

Compositions

The present invention also relates to compositions comprising apolypeptide of the present invention. Preferably, the compositions areenriched in such a polypeptide. The term “enriched” indicates that thecellobiohydrolase of the composition has been increased, e.g., with anenrichment factor of at least 1.1.

The compositions may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the compositions may comprise multiple enzymaticactivities, such as one or more (e.g., several) additional enzymesselected from the group consisting of a cellulase, a polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.

The compositions may be prepared in accordance with methods known in theart and may be in the form of a liquid or a dry composition. Thecompositions may be stabilized in accordance with methods known in theart.

In one embodiment, the composition further comprises one or more enzymesselected from the group consisting of one or more (e.g., several)xylanases, mannanases, glucanases, cellulases, lipases, esterases,proteases, endoglycosidases, endo-beta-1,4-glucanases, beta-glucanases,endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases,laccases, amylases, glucoamylases, pectinases, reductases, oxidases,phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases,mannanases, xyloglucanases, xylanases, mannanases, glucanases, pectinacetyl esterases, rhamnogalacturonan acetyl esterases,polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases,pectin methylesterases, and transglutaminases.

The compositions may be prepared in accordance with methods known in theart and may have any physical appearance such as liquid, paste or solid.For instance, the polypeptide composition may be formulated usingmethods known to the art of formulating enzymes and/or pharmaceuticalproducts, e.g., into coated or uncoated granules or micro granules. Thepolypeptide to be included in the composition may be stabilized inaccordance with methods known in the art, e.g., by stabilizing thepolypeptide in the composition by adding an antioxidant or reducingagent to limit oxidation or the polypeptide may be stabilized by addingpolymers such as PVP, PVA, PEG or other suitable polymers known to bebeneficial to the stability of polypeptides in solid or liquidcompositions.

The compositions may be a fermentation broth formulation or a cellcomposition, as described herein. Consequently, the present inventionalso relates to fermentation broth formulations and cell compositionscomprising a polypeptide having cellobiohydrolase, endoglucanase,cellulolytic enhancing, or xylanase activity of the present invention.In some embodiments, the composition is a cell-killed whole brothcontaining organic acid(s), killed cells and/or cell debris, and culturemedium.

The term “fermentation broth” as used herein refers to a preparationproduced by cellular fermentation that undergoes no or minimal recoveryand/or purification. For example, fermentation broths are produced whenmicrobial cultures are grown to saturation, incubated undercarbon-limiting conditions to allow protein synthesis (e.g., expressionof enzymes by host cells) and secretion into cell culture medium. Thefermentation broth can contain unfractionated or fractionated contentsof the fermentation materials derived at the end of the fermentation.Typically, the fermentation broth is unfractionated and comprises thespent culture medium and cell debris present after the microbial cells(e.g., filamentous fungal cells) are removed, e.g., by centrifugation.In some embodiments, the fermentation broth contains spent cell culturemedium, extracellular enzymes, and viable and/or nonviable microbialcells.

In an embodiment, the fermentation broth formulation and cellcompositions comprise a first organic acid component comprising at leastone 1-5 carbon organic acid and/or a salt thereof and a second organicacid component comprising at least one 6 or more carbon organic acidand/or a salt thereof. In a specific embodiment, the first organic acidcomponent is acetic acid, formic acid, propionic acid, a salt thereof,or a mixture of two or more of the foregoing and the second organic acidcomponent is benzoic acid, cyclohexanecarboxylic acid, 4-methylvalericacid, phenylacetic acid, a salt thereof, or a mixture of two or more ofthe foregoing.

In one aspect, the composition contains an organic acid(s), andoptionally further contains killed cells and/or cell debris. In oneembodiment, the killed cells and/or cell debris are removed from acell-killed whole broth to provide a composition that is free of thesecomponents.

The fermentation broth formulations or cell compostions may furthercomprise a preservative and/or anti-microbial (e.g., bacteriostatic)agent, including, but not limited to, sorbitol, sodium chloride,potassium sorbate, and others known in the art.

The cell-killed whole broth or composition may further comprise one ormore enzyme activities such as acetylxylan esterase,alpha-arabinofuranosidase, alpha-galactosidase, alpha-glucuronidase,amylase, arabinanase, arabinofuranosidase, beta-galactosidase,beta-glucosidase, cellobiohydrolase, endoglucanase,endo-beta-1,3(4)-glucanase, ferrulic acid esterase, galactanase,glucoamylase, glucohydrolase, hybrid peroxidases, with combinedproperties of lignin peroxidases and manganese-dependent peroxidases,laccase, lignin peroxidase, manganese-dependent peroxidases, mannanase,mannan acetyl esterase, mannosidase, pectate lyase, pectin acetylesterase, pectinase lyase, pectin methyl esterase, polygalacturonase,protease, rhamnogalacturonan lyase, rhamnogalacturonan acetyl esterase,rhamnogalacturonase, xylanase, xylogalacturonosidase, xylogalacturonase,xyloglucanase, and xylosidase.

In some embodiments, the cell-killed whole broth or composition includescellulolytic enzymes including, but not limited to, (i) endoglucanases(EG) or 1,4-D-glucan-4-glucanohydrolases (EC 3.2.1.4), (ii)exoglucanases, including 1,4-D-glucan glucanohydrolases (also known ascellodextnnases) (EC 3.2.1.74) and 1,4-D-glucan cellobiohydrolases(exo-cellobiohydrolases, CBH) (EC 3.2.1.91), and (iii) beta-glucosidase(BG) or beta-glucoside glucohydrolases (EC 3.2.1.21).

The cell-killed whole broth or composition may contain theunfractionated contents of the fermentation materials derived at the endof the fermentation. Typically, the cell-killed whole broth orcomposition contains the spent culture medium and cell debris presentafter the microbial cells (e.g., filamentous fungal cells) are grown tosaturation, incubated under carbon-limiting conditions to allow proteinsynthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)).In some embodiments, the cell-killed whole broth or composition containsthe spent cell culture medium, extracellular enzymes, and killedfilamentous fungal cells. In some embodiments, the microbial cellspresent in the cell-killed whole broth or composition can bepermeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically aliquid, but may contain insoluble components, such as killed cells, celldebris, culture media components, and/or insoluble enzyme(s). In someembodiments, insoluble components may be removed to provide a clarifiedliquid composition.

The whole broth formulations and cell compositions of the presentinvention may be produced by a method described in WO 90/15861 or WO2010/096673.

Examples are given below of preferred uses of the compositions of thepresent invention. The dosage of the composition and other conditionsunder which the composition is used may be determined on the basis ofmethods known in the art.

Uses

The present invention is also directed to the following methods forusing the polypeptides having cellobiohydrolase activity, orcompositions thereof.

The present invention also relates to methods for degrading a cellulosicmaterial, comprising: treating the cellulosic material with an enzymecomposition in the presence of a polypeptide having cellobiohydrolaseactivity of the present invention. In one aspect, the methods furthercomprise recovering the degraded or converted cellulosic material.Soluble products of degradation or conversion of the cellulosic materialcan be separated from insoluble cellulosic material using a method knownin the art such as, for example, centrifugation, filtration, or gravitysettling.

The present invention also relates to methods of producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition in the presence of a polypeptidehaving cellobiohydrolase activity of the present invention; (b)fermenting the saccharified cellulosic material with one or more (e.g.,several) fermenting microorganisms to produce the fermentation product;and (c) recovering the fermentation product from the fermentation.

The present invention also relates to methods of fermenting a cellulosicmaterial, comprising: fermenting the cellulosic material with one ormore (e.g., several) fermenting microorganisms, wherein the cellulosicmaterial is saccharified with an enzyme composition in the presence of apolypeptide having cellobiohydrolase activity of the present invention.In one aspect, the fermenting of the cellulosic material produces afermentation product. In another aspect, the methods further compriserecovering the fermentation product from the fermentation.

The methods of the present invention can be used to saccharify thecellulosic material to fermentable sugars and to convert the fermentablesugars to many useful fermentation products, e.g., fuel, potableethanol, and/or platform chemicals (e.g., acids, alcohols, ketones,gases, and the like). The production of a desired fermentation productfrom the cellulosic material typically involves pretreatment, enzymatichydrolysis (saccharification), and fermentation.

The processing of the cellulosic material according to the presentinvention can be accomplished using methods conventional in the art.Moreover, the methods of the present invention can be implemented usingany conventional biomass processing apparatus configured to operate inaccordance with the invention.

Hydrolysis (saccharification) and fermentation, separate orsimultaneous, include, but are not limited to, separate hydrolysis andfermentation (SHF); simultaneous saccharification and fermentation(SSF); simultaneous saccharification and co-fermentation (SSCF); hybridhydrolysis and fermentation (HHF); separate hydrolysis andco-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF);and direct microbial conversion (DMC), also sometimes calledconsolidated bioprocessing (CBP). SHF uses separate process steps tofirst enzymatically hydrolyze the cellulosic material to fermentablesugars, e.g., glucose, cellobiose, and pentose monomers, and thenferment the fermentable sugars to ethanol. In SSF, the enzymatichydrolysis of the cellulosic material and the fermentation of sugars toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF involves the co-fermentation of multiple sugars (Sheehanand Himmel, 1999, Enzymes, energy and the environment: A strategicperspective on the U.S. Department of Energy's research and developmentactivities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF involvesa separate hydrolysis step, and in addition a simultaneoussaccharification and hydrolysis step, which can be carried out in thesame reactor. The steps in an HHF process can be carried out atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(enzyme production, hydrolysis, and fermentation) in one or more (e.g.,several) steps where the same organism is used to produce the enzymesfor conversion of the cellulosic material to fermentable sugars and toconvert the fermentable sugars into a final product (Lynd et al., 2002,Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that anymethod known in the art comprising pretreatment, enzymatic hydrolysis(saccharification), fermentation, or a combination thereof, can be usedin the practicing the methods of the present invention.

A conventional apparatus can include a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor withultrafiltration, and/or a continuous plug-flow column reactor (Corazzaet al., 2003, Optimal control in fed-batch reactor for the cellobiosehydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov andSinitsyn, 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. Amathematical model for a batch reactor process, Enz. Microb. Technol. 7:346-352), an attrition reactor (Ryu and Lee, 1983, Bioconversion ofwaste cellulose by using an attrition bioreactor, Biotechnol. Bioeng.25: 53-65), or a reactor with intensive stirring induced by anelectromagnetic field (Gusakov et al., 1996, Enhancement of enzymaticcellulose hydrolysis using a novel type of bioreactor with intensivestirring induced by electromagnetic field, Appl. Biochem. Biotechnol.56: 141-153). Additional reactor types include fluidized bed, upflowblanket, immobilized, and extruder type reactors for hydrolysis and/orfermentation.

Pretreatment.

In practicing the methods of the present invention, any pretreatmentprocess known in the art can be used to disrupt plant cell wallcomponents of the cellulosic material (Chandra et al., 2007, Substratepretreatment: The key to effective enzymatic hydrolysis oflignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbeand Zacchi, 2007, Pretreatment of lignocellulosic materials forefficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance thedigestibility of lignocellulosic biomass, Bioresource Technol. 100:10-18; Mosier et al., 2005, Features of promising technologies forpretreatment of lignocellulosic biomass, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosicwastes to improve ethanol and biogas production: A review, Int. J. ofMol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key tounlocking low-cost cellulosic ethanol, Biofuels Bioproducts andBiorefining-Biofpr. 2: 26-40).

The cellulosic material can also be subjected to particle sizereduction, sieving, pre-soaking, wetting, washing, and/or conditioningprior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steampretreatment (with or without explosion), dilute acid pretreatment, hotwater pretreatment, alkaline pretreatment, lime pretreatment, wetoxidation, wet explosion, ammonia fiber explosion, organosolvpretreatment, and biological pretreatment. Additional pretreatmentsinclude ammonia percolation, ultrasound, electroporation, microwave,supercritical CO₂, supercritical H₂O, ozone, ionic liquid, and gammairradiation pretreatments.

The cellulosic material can be pretreated before hydrolysis and/orfermentation. Pretreatment is preferably performed prior to thehydrolysis. Alternatively, the pretreatment can be carried outsimultaneously with enzyme hydrolysis to release fermentable sugars,such as glucose, xylose, and/or cellobiose. In most cases thepretreatment step itself results in some conversion of biomass tofermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the cellulosic material isheated to disrupt the plant cell wall components, including lignin,hemicellulose, and cellulose to make the cellulose and other fractions,e.g., hemicellulose, accessible to enzymes. The cellulosic material ispassed to or through a reaction vessel where steam is injected toincrease the temperature to the required temperature and pressure and isretained therein for the desired reaction time. Steam pretreatment ispreferably performed at 140-250° C., e.g., 160-200° C. or 170-190° C.,where the optimal temperature range depends on addition of a chemicalcatalyst. Residence time for the steam pretreatment is preferably 1-60minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10minutes, where the optimal residence time depends on temperature rangeand addition of a chemical catalyst. Steam pretreatment allows forrelatively high solids loadings, so that the cellulosic material isgenerally only moist during the pretreatment. The steam pretreatment isoften combined with an explosive discharge of the material after thepretreatment, which is known as steam explosion, that is, rapid flashingto atmospheric pressure and turbulent flow of the material to increasethe accessible surface area by fragmentation (Duff and Murray, 1996,Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl.Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No.20020164730). During steam pretreatment, hemicellulose acetyl groups arecleaved and the resulting acid autocatalyzes partial hydrolysis of thehemicellulose to monosaccharides and oligosaccharides. Lignin is removedto only a limited extent.

Chemical Pretreatment: The term “chemical treatment” refers to anychemical pretreatment that promotes the separation and/or release ofcellulose, hemicellulose, and/or lignin. Such a pretreatment can convertcrystalline cellulose to amorphous cellulose. Examples of suitablechemical pretreatment processes include, for example, dilute acidpretreatment, lime pretreatment, wet oxidation, ammonia fiber/freezeexplosion (AFEX), ammonia percolation (APR), ionic liquid, andorganosolv pretreatments.

A catalyst such as H₂SO₄ or SO₂ (typically 0.3 to 5% w/w) is often addedprior to steam pretreatment, which decreases the time and temperature,increases the recovery, and improves enzymatic hydrolysis (Ballesteroset al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al.,2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006,Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, thecellulosic material is mixed with dilute acid, typically H₂SO₄, andwater to form a slurry, heated by steam to the desired temperature, andafter a residence time flashed to atmospheric pressure. The dilute acidpretreatment can be performed with a number of reactor designs, e.g.,plug-flow reactors, counter-current reactors, or continuouscounter-current shrinking bed reactors (Duff and Murray, 1996, supra;Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999,Adv. Biochem. Eng. Biotechnol. 65: 93-115).

Several methods of pretreatment under alkaline conditions can also beused. These alkaline pretreatments include, but are not limited to,sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), andammonia fiber/freeze explosion (AFEX).

Lime pretreatment is performed with calcium oxide or calcium hydroxideat temperatures of 85-150° C. and residence times from 1 hour to severaldays (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier etal., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatmentmethods using ammonia.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C. for 5-15 minutes with addition of an oxidative agent such as hydrogenperoxide or over-pressure of oxygen (Schmidt and Thomsen, 1998,Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem.Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88:567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669-1677). The pretreatment is performed preferably at 1-40% drymatter, e.g., 2-30% dry matter or 5-20% dry matter, and often theinitial pH is increased by the addition of alkali such as sodiumcarbonate.

A modification of the wet oxidation pretreatment method, known as wetexplosion (combination of wet oxidation and steam explosion) can handledry matter up to 30%. In wet explosion, the oxidizing agent isintroduced during pretreatment after a certain residence time. Thepretreatment is then ended by flashing to atmospheric pressure (WO2006/032282).

Ammonia fiber explosion (AFEX) involves treating the cellulosic materialwith liquid or gaseous ammonia at moderate temperatures such as 90-150°C. and high pressure such as 17-20 bar for 5-10 minutes, where the drymatter content can be as high as 60% (Gollapalli et al., 2002, Appl.Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol.Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol.121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96:2014-2018). During AFEX pretreatment cellulose and hemicelluloses remainrelatively intact. Lignin-carbohydrate complexes are cleaved.

Organosolv pretreatment delignifies the cellulosic material byextraction using aqueous ethanol (40-60% ethanol) at 160-200° C. for30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan etal., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl.Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as acatalyst. In organosolv pretreatment, the majority of hemicellulose andlignin is removed.

Other examples of suitable pretreatment methods are described by Schellet al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, andMosier et al., 2005, Bioresource Technology 96: 673-686, and U.S.Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as adilute acid treatment, and more preferably as a continuous dilute acidtreatment. The acid is typically sulfuric acid, but other acids can alsobe used, such as acetic acid, citric acid, nitric acid, phosphoric acid,tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof.Mild acid treatment is conducted in the pH range of preferably 1-5,e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in therange from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or0.1 to 2 wt % acid. The acid is contacted with the cellulosic materialand held at a temperature in the range of preferably 140-200° C., e.g.,165-190° C., for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. Inpreferred aspects, the cellulosic material is present duringpretreatment in amounts preferably between 10-80 wt %, e.g., 20-70 wt %or 30-60 wt %, such as around 40 wt %. The pretreated cellulosicmaterial can be unwashed or washed using any method known in the art,e.g., washed with water.

Mechanical Pretreatment or Physical Pretreatment: The term “mechanicalpretreatment” or “physical pretreatment” refers to any pretreatment thatpromotes size reduction of particles. For example, such pretreatment caninvolve various types of grinding or milling (e.g., dry milling, wetmilling, or vibratory ball milling).

The cellulosic material can be pretreated both physically (mechanically)and chemically. Mechanical or physical pretreatment can be coupled withsteaming/steam explosion, hydrothermolysis, dilute or mild acidtreatment, high temperature, high pressure treatment, irradiation (e.g.,microwave irradiation), or combinations thereof. In one aspect, highpressure means pressure in the range of preferably about 100 to about400 psi, e.g., about 150 to about 250 psi. In another aspect, hightemperature means temperatures in the range of about 100 to about 300°C., e.g., about 140 to about 200° C. In a preferred aspect, mechanicalor physical pretreatment is performed in a batch-process using a steamgun hydrolyzer system that uses high pressure and high temperature asdefined above, e.g., a Sunds Hydrolyzer available from Sunds DefibratorAB, Sweden. The physical and chemical pretreatments can be carried outsequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the cellulosic material is subjectedto physical (mechanical) or chemical pretreatment, or any combinationthereof, to promote the separation and/or release of cellulose,hemicellulose, and/or lignin.

Biological Pretreatment: The term “biological pretreatment” refers toany biological pretreatment that promotes the separation and/or releaseof cellulose, hemicellulose, and/or lignin from the cellulosic material.Biological pretreatment techniques can involve applyinglignin-solubilizing microorganisms and/or enzymes (see, for example,Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol:Production and Utilization, Wyman, C. E., ed., Taylor & Francis,Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical andbiological treatments for enzymatic/microbial conversion of cellulosicbiomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994,Pretreating lignocellulosic biomass: a review, in Enzymatic Conversionof Biomass for Fuels Production, Himmel, M. E., Baker, J. O., andOverend, R. P., eds., ACS Symposium Series 566, American ChemicalSociety, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J.,and Tsao, G. T., 1999, Ethanol production from renewable resources, inAdvances in Biochemical Engineering/Biotechnology, Scheper, T., ed.,Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson andHahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates forethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander andEriksson, 1990, Production of ethanol from lignocellulosic materials:State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification.

In the hydrolysis step, also known as saccharification, the cellulosicmaterial, e.g., pretreated, is hydrolyzed to break down cellulose and/orhemicellulose to fermentable sugars, such as glucose, cellobiose,xylose, xylulose, arabinose, mannose, galactose, and/or solubleoligosaccharides. The hydrolysis is performed enzymatically by an enzymecomposition in the presence of a polypeptide having cellobiohydrolaseactivity of the present invention. The enzymes of the compositions canbe added simultaneously or sequentially.

Enzymatic hydrolysis is preferably carried out in a suitable aqueousenvironment under conditions that can be readily determined by oneskilled in the art. In one aspect, hydrolysis is performed underconditions suitable for the activity of the enzyme(s), i.e., optimal forthe enzyme(s). The hydrolysis can be carried out as a fed batch orcontinuous process where the cellulosic material is fed gradually to,for example, an enzyme containing hydrolysis solution.

The saccharification is generally performed in stirred-tank reactors orfermentors under controlled pH, temperature, and mixing conditions.Suitable process time, temperature and pH conditions can readily bedetermined by one skilled in the art. For example, the saccharificationcan last up to 200 hours, but is typically performed for preferablyabout 12 to about 120 hours, e.g., about 16 to about 72 hours or about24 to about 48 hours. The temperature is in the range of preferablyabout 25° C. to about 70° C., e.g., about 30° C. to about 65° C., about40° C. to about 60° C., or about 50° C. to about 55° C. The pH is in therange of preferably about 3 to about 8, e.g., about 3.5 to about 7,about 4 to about 6, or about 5.0 to about 5.5. The dry solids content isin the range of preferably about 5 to about 50 wt %, e.g., about 10 toabout 40 wt % or about 20 to about 30 wt %.

The enzyme compositions can comprise any protein useful in degrading thecellulosic material.

In one aspect, the enzyme composition comprises or further comprises oneor more (e.g., several) proteins selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin. Inanother aspect, the cellulase is preferably one or more (e.g., several)enzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase. In another aspect, thehemicellulase is preferably one or more (e.g., several) enzymes selectedfrom the group consisting of an acetylmannan esterase, an acetylxylanesterase, an arabinanase, an arabinofuranosidase, a coumaric acidesterase, a feruloyl esterase, a galactosidase, a glucuronidase, aglucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and axylosidase.

In another aspect, the enzyme composition comprises one or more (e.g.,several) cellulolytic enzymes. In another aspect, the enzyme compositioncomprises or further comprises one or more (e.g., several)hemicellulolytic enzymes. In another aspect, the enzyme compositioncomprises one or more (e.g., several) cellulolytic enzymes and one ormore (e.g., several) hemicellulolytic enzymes. In another aspect, theenzyme composition comprises one or more (e.g., several) enzymesselected from the group of cellulolytic enzymes and hemicellulolyticenzymes. In another aspect, the enzyme composition comprises anendoglucanase. In another aspect, the enzyme composition comprises acellobiohydrolase. In another aspect, the enzyme composition comprises abeta-glucosidase. In another aspect, the enzyme composition comprises apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase and a polypeptidehaving cellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a cellobiohydrolase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises a beta-glucosidase and a polypeptide havingcellulolytic enhancing activity. In another aspect, the enzymecomposition comprises an endoglucanase and a cellobiohydrolase. Inanother aspect, the enzyme composition comprises an endoglucanase and abeta-glucosidase. In another aspect, the enzyme composition comprises acellobiohydrolase and a beta-glucosidase. In another aspect, the enzymecomposition comprises an endoglucanase, a cellobiohydrolase, and apolypeptide having cellulolytic enhancing activity. In another aspect,the enzyme composition comprises an endoglucanase, a beta-glucosidase,and a polypeptide having cellulolytic enhancing activity. In anotheraspect, the enzyme composition comprises a cellobiohydrolase, abeta-glucosidase, and a polypeptide having cellulolytic enhancingactivity. In another aspect, the enzyme composition comprises anendoglucanase, a cellobiohydrolase, and a beta-glucosidase. In anotheraspect, the enzyme composition comprises an endoglucanase, acellobiohydrolase, a beta-glucosidase, and a polypeptide havingcellulolytic enhancing activity.

In another aspect, the enzyme composition comprises an acetylmannanesterase. In another aspect, the enzyme composition comprises anacetylxylan esterase. In another aspect, the enzyme compositioncomprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect,the enzyme composition comprises an arabinofuranosidase (e.g.,alpha-L-arabinofuranosidase). In another aspect, the enzyme compositioncomprises a coumaric acid esterase. In another aspect, the enzymecomposition comprises a feruloyl esterase. In another aspect, the enzymecomposition comprises a galactosidase (e.g., alpha-galactosidase and/orbeta-galactosidase). In another aspect, the enzyme composition comprisesa glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, theenzyme composition comprises a glucuronoyl esterase. In another aspect,the enzyme composition comprises a mannanase. In another aspect, theenzyme composition comprises a mannosidase (e.g., beta-mannosidase). Inanother aspect, the enzyme composition comprises a xylanase. In apreferred aspect, the xylanase is a Family 10 xylanase. In anotheraspect, the enzyme composition comprises a xylosidase (e.g.,beta-xylosidase).

In another aspect, the enzyme composition comprises an esterase. Inanother aspect, the enzyme composition comprises an expansin. In anotheraspect, the enzyme composition comprises a laccase. In another aspect,the enzyme composition comprises a ligninolytic enzyme. In a preferredaspect, the ligninolytic enzyme is a manganese peroxidase. In anotherpreferred aspect, the ligninolytic enzyme is a lignin peroxidase. Inanother preferred aspect, the ligninolytic enzyme is a H₂O₂-producingenzyme. In another aspect, the enzyme composition comprises a pectinase.In another aspect, the enzyme composition comprises a peroxidase. Inanother aspect, the enzyme composition comprises a protease. In anotheraspect, the enzyme composition comprises a swollenin.

In the methods of the present invention, the enzyme(s) can be addedprior to or during saccharification, saccharification and fermentation,or fermentation.

One or more (e.g., several) components of the enzyme composition may bewild-type proteins, recombinant proteins, or a combination of wild-typeproteins and recombinant proteins. For example, one or more (e.g.,several) components may be native proteins of a cell, which is used as ahost cell to express recombinantly one or more (e.g., several) othercomponents of the enzyme composition. One or more (e.g., several)components of the enzyme composition may be produced as monocomponents,which are then combined to form the enzyme composition. The enzymecomposition may be a combination of multicomponent and monocomponentprotein preparations.

The enzymes used in the methods of the present invention may be in anyform suitable for use, such as, for example, a fermentation brothformulation or a cell composition, a cell lysate with or withoutcellular debris, a semi-purified or purified enzyme preparation, or ahost cell as a source of the enzymes. The enzyme composition may be adry powder or granulate, a non-dusting granulate, a liquid, a stabilizedliquid, or a stabilized protected enzyme. Liquid enzyme preparationsmay, for instance, be stabilized by adding stabilizers such as a sugar,a sugar alcohol or another polyol, and/or lactic acid or another organicacid according to established processes.

The optimum amounts of the enzymes and polypeptides havingcellobiohydrolase activity depend on several factors including, but notlimited to, the mixture of component cellulolytic enzymes and/orhemicellulolytic enzymes, the cellulosic material, the concentration ofcellulosic material, the pretreatment(s) of the cellulosic material,temperature, time, pH, and inclusion of fermenting organism (e.g., yeastfor Simultaneous Saccharification and Fermentation).

In one aspect, an effective amount of cellulolytic or hemicellulolyticenzyme to the cellulosic material is about 0.5 to about 50 mg, e.g.,about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5to about 10 mg per g of the cellulosic material.

In another aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to the cellulosic material is about 0.01 toabout 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 toabout 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg,about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 toabout 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosicmaterial.

In another aspect, an effective amount of a polypeptide havingcellobiohydrolase activity to cellulolytic or hemicellulolytic enzyme isabout 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g,about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g ofcellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolyticenzyme activity as well as other proteins/polypeptides useful in thedegradation of the cellulosic material, e.g., GH61 polypeptides havingcellulolytic enhancing activity (collectively hereinafter “polypeptideshaving enzyme activity”) can be derived or obtained from any suitableorigin, including, bacterial, fungal, yeast, plant, or mammalian origin.The term “obtained” also means herein that the enzyme may have beenproduced recombinantly in a host organism employing methods describedherein, wherein the recombinantly produced enzyme is either native orforeign to the host organism or has a modified amino acid sequence,e.g., having one or more (e.g., several) amino acids that are deleted,inserted and/or substituted, i.e., a recombinantly produced enzyme thatis a mutant and/or a fragment of a native amino acid sequence or anenzyme produced by nucleic acid shuffling processes known in the art.Encompassed within the meaning of a native enzyme are natural variantsand within the meaning of a foreign enzyme are variants obtainedrecombinantly, such as by site-directed mutagenesis or shuffling.

A polypeptide having enzyme activity may be a bacterial polypeptide. Forexample, the polypeptide may be a Gram positive bacterial polypeptidesuch as a Bacillus, Streptococcus, Streptomyces, Staphylococcus,Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus,Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacilluspolypeptide having enzyme activity, or a Gram negative bacterialpolypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, orUreaplasma polypeptide having enzyme activity.

In one aspect, the polypeptide is a Bacillus alkalophilus, Bacillusamyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillusclausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacilluslentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus,Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptococcus equisimilis,Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equisubsp. Zooepidemicus polypeptide having enzyme activity.

In another aspect, the polypeptide is a Streptomyces achromogenes,Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus,or Streptomyces lividans polypeptide having enzyme activity.

The polypeptide having enzyme activity may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having enzyme activity; or more preferably a filamentousfungal polypeptide such as an Acremonium, Agaricus, Alternaria,Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium,Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes,Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium,Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula,Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor,Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium,Phanerochaete, Piromyces, Poitrasia, Pseudoplectania,Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces,Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea,Verticillium, Volvariella, or Xylaria polypeptide having enzymeactivity.

In one aspect, the polypeptide is a Saccharomyces carlsbergensis,Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomycesdouglasfi, Saccharomyces kluyveri, Saccharomyces norbensis, orSaccharomyces oviformis polypeptide having enzyme activity.

In another aspect, the polypeptide is an Acremonium cellulolyticus,Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus,Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum,Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporiummerdarium, Chrysosporium mops, Chrysosporium pannicola, Chrysosporiumqueenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa,Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurosporacrassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaetechrysosporium, Thielavia achromatica, Thielavia albomyces, Thielaviaalbopilosa, Thielavia australeinsis, Thielavia fimeti, Thielaviamicrospora, Thielavia ovispora, Thielavia peruviana, Thielaviaspededonium, Thielavia setosa, Thielavia subthermophila, Thielaviaterrestris, Trichoderma harzianum, Trichoderma koningfi, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaeasaccata polypeptide having enzyme activity.

Chemically modified or protein engineered mutants of polypeptides havingenzyme activity may also be used.

One or more (e.g., several) components of the enzyme composition may bea recombinant component, i.e., produced by cloning of a DNA sequenceencoding the single component and subsequent cell transformed with theDNA sequence and expressed in a host (see, for example, WO 91/17243 andWO 91/17244). The host is preferably a heterologous host (enzyme isforeign to host), but the host may under certain conditions also be ahomologous host (enzyme is native to host). Monocomponent cellulolyticproteins may also be prepared by purifying such a protein from afermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymescomprise a commercial cellulolytic enzyme preparation. Examples ofcommercial cellulolytic enzyme preparations suitable for use in thepresent invention include, for example, CELLIC® CTec (Novozymes A/S),CELLIC® CTec2 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188(Novozymes A/S), CELLUZYME™ (Novozymes A/S), CEREFLO™ (Novozymes A/S),and ULTRAFLO™ (Novozymes A/S), ACCELERASE™ (Genencor Int.), LAMINEX™(Genencor Int.), SPEZYME™ CP (Genencor Int.), FILTRASE® NL (DSM);METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (ROhm GmbH), FIBREZYME® LDI(Dyadic International, Inc.), FIBREZYME® LBR (Dyadic International,Inc.), or VISCOSTAR® 150L (Dyadic International, Inc.). The cellulaseenzymes are added in amounts effective from about 0.001 to about 5.0 wt% of solids, e.g., about 0.025 to about 4.0 wt % of solids or about0.005 to about 2.0 wt % of solids.

Examples of bacterial endoglucanases that can be used in the methods ofthe present invention, include, but are not limited to, an Acidothermuscellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No.5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); andThermobifida fusca endoglucanase V (WO 05/093050).

Examples of fungal endoglucanases that can be used in the presentinvention, include, but are not limited to, a Trichoderma reeseiendoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichodermareesei Cel7B endoglucanase I (GENBANK™ accession no. M15665),Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK™ accessionno. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988,Appl. Environ. Microbiol. 64: 555-563, GENBANK™ accession no. AB003694),Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, MolecularMicrobiology 13: 219-228, GENBANK™ accession no. Z33381), Aspergillusaculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18:5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995,Current Genetics 27: 435-439), Erwinia carotovara endoglucanase(Saarilahti et al., 1990, Gene 90: 9-14), Fusarium oxysporumendoglucanase (GENBANK™ accession no. L29381), Humicola grisea var.thermoidea endoglucanase (GENBANK™ accession no. AB003107), Melanocarpusalbomyces endoglucanase (GENBANK™ accession no. MAL515703), Neurosporacrassa endoglucanase (GENBANK™ accession no. XM_324477), Humicolainsolens endoglucanase V, Myceliophthora thermophila CBS 117.65endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6Bendoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase,Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestrisNRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7Fendoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7Aendoglucanase, and Trichoderma reesei strain No. VTT-D-80133endoglucanase (GENBANK™ accession no. M15665).

Examples of cellobiohydrolases useful in the present invention include,but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomiumthermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolaseI, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871),Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielaviaterrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichodermareesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, andTrichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include,but are not limited to, beta-glucosidases from Aspergillus aculeatus(Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275:4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianumIBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (\NO2011/035029), and Trichophaea saccata (WO 2007/019442).

The beta-glucosidase may be a fusion protein. In one aspect, thebeta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BGfusion protein (WO 2008/057637) or an Aspergillus oryzaebeta-glucosidase fusion protein (WO 2008/057637).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidasesare disclosed in numerous Glycosyl Hydrolase families using theclassification according to Henrissat, 1991, A classification ofglycosyl hydrolases based on amino-acid sequence similarities, Biochem.J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating thesequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

Other cellulolytic enzymes that may be used in the present invention aredescribed in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO99/10481, WO 99/025847, WO 99/031255, WO 2002/101078, WO 2003/027306, WO2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO2008/008070, WO 2008/008793, U.S. Pat. Nos. 5,457,046, 5,648,263, and5,686,593.

In the methods of the present invention, any GH61 polypeptide havingcellulolytic enhancing activity can be used as a component of the enzymecomposition.

Examples of GH61 polypeptides having cellulolytic enhancing activityuseful in the methods of the present invention include, but are notlimited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647,WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290),Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754),GH61 polypeptides from Penicillium pinophilum (WO 2011/005867),Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397), andThermoascus crustaceous (WO 2011/041504).

In one aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a soluble activating divalent metalcation according to WO 2008/151043, e.g., manganese or copper sulfate.

In another aspect, the GH61 polypeptide having cellulolytic enhancingactivity is used in the presence of a dioxy compound, a bicyliccompound, a heterocyclic compound, a nitrogen-containing compound, aquinone compound, a sulfur-containing compound, or a liquor obtainedfrom a pretreated cellulosic material such as pretreated corn stover(PCS).

The dioxy compound may include any suitable compound containing two ormore oxygen atoms. In some aspects, the dioxy compounds contain asubstituted aryl moiety as described herein. The dioxy compounds maycomprise one or more (e.g., several) hydroxyl and/or hydroxylderivatives, but also include substituted aryl moieties lacking hydroxyland hydroxyl derivatives. Non-limiting examples of the dioxy compoundsinclude pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoicacid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid;methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone;2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid;4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethylgallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol;(croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol;3-ethyoxy-1,2-propanediol; 2,4,4′-trihydroxybenzophenone;cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione;dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate;4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or asalt or solvate thereof.

The bicyclic compound may include any suitable substituted fused ringsystem as described herein. The compounds may comprise one or more(e.g., several) additional rings, and are not limited to a specificnumber of rings unless otherwise stated. In one aspect, the bicycliccompound is a flavonoid. In another aspect, the bicyclic compound is anoptionally substituted isoflavonoid. In another aspect, the bicycliccompound is an optionally substituted flavylium ion, such as anoptionally substituted anthocyanidin or optionally substitutedanthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin;kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin;cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

The heterocyclic compound may be any suitable compound, such as anoptionally substituted aromatic or non-aromatic ring comprising aheteroatom, as described herein. In one aspect, the heterocyclic is acompound comprising an optionally substituted heterocycloalkyl moiety oran optionally substituted heteroaryl moiety. In another aspect, theoptionally substituted heterocycloalkyl moiety or optionally substitutedheteroaryl moiety is an optionally substituted 5-memberedheterocycloalkyl or an optionally substituted 5-membered heteroarylmoiety. In another aspect, the optionally substituted heterocycloalkylor optionally substituted heteroaryl moiety is an optionally substitutedmoiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl,oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl,thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl,carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl,quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl,benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl,dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl,pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl,piperidinyl, and oxepinyl. In another aspect, the optionally substitutedheterocycloalkyl moiety or optionally substituted heteroaryl moiety isan optionally substituted furanyl. Non-limiting examples of theheterocyclic compounds include(1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one;4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone;[1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; α-hydroxy-γ-butyrolactone;ribonic γ-lactone; aldohexuronicaldohexuronic acid γ-lactone; gluconicacid δ-lactone; 4-hydroxycoumarin; dihydrobenzofuran;5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone;5,6-dihydro-2H-pyran-2-one; and5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvatethereof.

The nitrogen-containing compound may be any suitable compound with oneor more nitrogen atoms. In one aspect, the nitrogen-containing compoundcomprises an amine, imine, hydroxylamine, or nitroxide moiety.Non-limiting examples of the nitrogen-containing compounds includeacetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol;1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy;5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; andmaleamic acid; or a salt or solvate thereof.

The quinone compound may be any suitable compound comprising a quinonemoiety as described herein. Non-limiting examples of the quinonecompounds include 1,4-benzoquinone; 1,4-naphthoquinone;2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone orcoenzyme Q₀; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone;1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione oradrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinolinequinone; or a salt or solvate thereof.

The sulfur-containing compound may be any suitable compound comprisingone or more sulfur atoms. In one aspect, the sulfur-containing comprisesa moiety selected from thionyl, thioether, sulfinyl, sulfonyl,sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limitingexamples of the sulfur-containing compounds include ethanethiol;2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid;benzenethiol; benzene-12-dithiol; cysteine; methionine; glutathione;cystine; or a salt or solvate thereof.

In one aspect, an effective amount of such a compound described above tocellulosic material as a molar ratio to glucosyl units of cellulose isabout 10⁻⁶ to about 10, e.g., about 10⁻⁶ to about 7.5, about 10⁻⁶ toabout 5, about 10⁻⁶ to about 2.5, about 10⁻⁶ to about 1, about 10⁻⁶ toabout 1, about 10⁻⁶ to about 10⁻¹, about 10⁻⁴ to about 10⁻¹, about 10⁻³to about 10⁻¹, or about 10⁻³ to about 10⁻². In another aspect, aneffective amount of such a compound described above is about 0.1 μM toabout 1 M, e.g., about 0.5 μM to about 0.75 M, about 0.75 μM to about0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μMto about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM,about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM toabout 1 mM.

The term “liquor” means the solution phase, either aqueous, organic, ora combination thereof, arising from treatment of a lignocellulose and/orhemicellulose material in a slurry, or monosaccharides thereof, e.g.,xylose, arabinose, mannose, etc., under conditions as described herein,and the soluble contents thereof. A liquor for cellulolytic enhancementof a GH61 polypeptide can be produced by treating a lignocellulose orhemicellulose material (or feedstock) by applying heat and/or pressure,optionally in the presence of a catalyst, e.g., acid, optionally in thepresence of an organic solvent, and optionally in combination withphysical disruption of the material, and then separating the solutionfrom the residual solids. Such conditions determine the degree ofcellulolytic enhancement obtainable through the combination of liquorand a GH61 polypeptide during hydrolysis of a cellulosic substrate by acellulase preparation. The liquor can be separated from the treatedmaterial using a method standard in the art, such as filtration,sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about10⁻⁶ to about 10 g per g of cellulose, e.g., about 10⁻⁶ to about 7.5 g,about 10⁻⁶ to about 5, about 10⁻⁶ to about 2.5 g, about 10⁻⁶ to about 1g, about 10⁻⁵ to about 1 g, about 10⁻⁵ to about 10⁻¹ g, about 10⁻⁴ toabout 10⁻¹ g, about 10⁻³ to about 10⁻¹ g, or about 10⁻³ to about 10⁻² gper g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymescomprise a commercial hemicellulolytic enzyme preparation. Examples ofcommercial hemicellulolytic enzyme preparations suitable for use in thepresent invention include, for example, SHEARZYME™ (Novozymes A/S),CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), VISCOZYME®(Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (NovozymesA/S), MULTI FECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor),ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (BiocatalystsLimit, Wales, UK).

Examples of xylanases useful in the methods of the present inventioninclude, but are not limited to, xylanases from Aspergillus aculeatus(GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp.(WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), andTrichophaea saccata GH10 (WO 2011/057083).

Examples of beta-xylosidases useful in the methods of the presentinvention include, but are not limited to, beta-xylosidases fromNeurospora crassa (SwissProt accession number Q7SOW4), Trichodermareesei (UniProtKB/TrEMBL accession number Q92458), and Talaromycesemersonii (SwissProt accession number Q8X212).

Examples of acetylxylan esterases useful in the methods of the presentinvention include, but are not limited to, acetylxylan esterases fromAspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprotaccession number Q2GVVX4), Chaetomium gracile (GeneSeqP accession numberAAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocreajecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880),Neurospora crassa (UniProt accession number q75259), Phaeosphaerianodorum (Uniprot accession number Q0UHJ1), and Thielavia terrestris NRRL8126 (WO 2009/042846).

Examples of feruloyl esterases (ferulic acid esterases) useful in themethods of the present invention include, but are not limited to,feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122),Neosartorya fischeri (UniProt Accession number A1D9T4), Neurosporacrassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum(WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO2010/065448).

Examples of arabinofuranosidases useful in the methods of the presentinvention include, but are not limited to, arabinofuranosidases fromAspergillus niger (GeneSeqP accession number AAR94170), Humicolainsolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus(WO 2006/114094).

Examples of alpha-glucuronidases useful in the methods of the presentinvention include, but are not limited to, alpha-glucuronidases fromAspergillus clavatus (UniProt accession number alcc12), Aspergillusfumigatus (SwissProt accession number Q4VWV45), Aspergillus niger(Uniprot accession number Q96VVX9), Aspergillus terreus (SwissProtaccession number Q0CJP9), Humicola insolens (WO 2010/014706),Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii(UniProt accession number Q8X211), and Trichoderma reesei (Uniprotaccession number Q99024).

The polypeptides having enzyme activity used in the methods of thepresent invention may be produced by fermentation of the above-notedmicrobial strains on a nutrient medium containing suitable carbon andnitrogen sources and inorganic salts, using procedures known in the art(see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A, 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand enzyme production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of an enzyme or protein. Fermentation may,therefore, be understood as comprising shake flask cultivation, orsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe enzyme to be expressed or isolated. The resulting enzymes producedby the methods described above may be recovered from the fermentationmedium and purified by conventional procedures.

Fermentation.

The fermentable sugars obtained from the hydrolyzed cellulosic materialcan be fermented by one or more (e.g., several) fermentingmicroorganisms capable of fermenting the sugars directly or indirectlyinto a desired fermentation product. “Fermentation” or “fermentationprocess” refers to any fermentation process or any process comprising afermentation step. Fermentation processes also include fermentationprocesses used in the consumable alcohol industry (e.g., beer and wine),dairy industry (e.g., fermented dairy products), leather industry, andtobacco industry. The fermentation conditions depend on the desiredfermentation product and fermenting organism and can easily bedetermined by one skilled in the art.

In the fermentation step, sugars, released from the cellulosic materialas a result of the pretreatment and enzymatic hydrolysis steps, arefermented to a product, e.g., ethanol, by a fermenting organism, such asyeast. Hydrolysis (saccharification) and fermentation can be separate orsimultaneous, as described herein.

Any suitable hydrolyzed cellulosic material can be used in thefermentation step in practicing the present invention. The material isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art.

The term “fermentation medium” is understood herein to refer to a mediumbefore the fermenting microorganism(s) is(are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism, includingbacterial and fungal organisms, suitable for use in a desiredfermentation process to produce a fermentation product. The fermentingorganism can be hexose and/or pentose fermenting organisms, or acombination thereof. Both hexose and pentose fermenting organisms arewell known in the art. Suitable fermenting microorganisms are able toferment, i.e., convert, sugars, such as glucose, xylose, xylulose,arabinose, maltose, mannose, galactose, and/or oligosaccharides,directly or indirectly into the desired fermentation product. Examplesof bacterial and fungal fermenting organisms producing ethanol aredescribed by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

Examples of fermenting microorganisms that can ferment hexose sugarsinclude bacterial and fungal organisms, such as yeast. Preferred yeastincludes strains of Candida, Kluyveromyces, and Saccharomyces, e.g.,Candida sonorensis, Kluyveromyces mancianus, and Saccharomycescerevisiae.

Examples of fermenting organisms that can ferment pentose sugars intheir native state include bacterial and fungal organisms, such as someyeast. Preferred xylose fermenting yeast include strains of Candida,preferably C. sheatae or C. sonorensis; and strains of Pichia,preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentosefermenting yeast include strains of Pachysolen, preferably P.tannophilus. Organisms not capable of fermenting pentose sugars, such asxylose and arabinose, may be genetically modified to do so by methodsknown in the art.

Examples of bacteria that can efficiently ferment hexose and pentose toethanol include, for example, Bacillus coagulans, Clostridiumacetobutylicum, Clostridium thermocellum, Clostridium phytofermentans,Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonasmobilis (Philippidis, 1996, supra).

Other fermenting organisms include strains of Bacillus, such as Bacilluscoagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C.entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis,and C. scehatae; Clostridium, such as C. acetobutylicum, C.thermocellum, and C. phytofermentans; E. coli, especially E. colistrains that have been genetically modified to improve the yield ofethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala;Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. mancianus, K.lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such asS. pombe; Thermoanaerobacter, such as Thermoanaerobactersaccharolyticum; and Zymomonas, such as Zymomonas mobilis.

In a preferred aspect, the yeast is a Bretannomyces. In a more preferredaspect, the yeast is Bretannomyces clausenii. In another preferredaspect, the yeast is a Candida. In another more preferred aspect, theyeast is Candida sonorensis. In another more preferred aspect, the yeastis Candida boidinii. In another more preferred aspect, the yeast isCandida blankii. In another more preferred aspect, the yeast is Candidabrassicae. In another more preferred aspect, the yeast is Candidadiddensii. In another more preferred aspect, the yeast is Candidaentomophiliia. In another more preferred aspect, the yeast is Candidapseudotropicalis. In another more preferred aspect, the yeast is Candidascehatae. In another more preferred aspect, the yeast is Candida utilis.In another preferred aspect, the yeast is a Clavispora. In another morepreferred aspect, the yeast is Clavispora lusitaniae. In another morepreferred aspect, the yeast is Clavispora opuntiae. In another preferredaspect, the yeast is a Kluyveromyces. In another more preferred aspect,the yeast is Kluyveromyces fragilis. In another more preferred aspect,the yeast is Kluyveromyces mancianus. In another more preferred aspect,the yeast is Kluyveromyces thermotolerans. In another preferred aspect,the yeast is a Pachysolen. In another more preferred aspect, the yeastis Pachysolen tannophilus. In another preferred aspect, the yeast is aPichia. In another more preferred aspect, the yeast is a Pichiastipitis. In another preferred aspect, the yeast is a Saccharomyces spp.In another more preferred aspect, the yeast is Saccharomyces cerevisiae.In another more preferred aspect, the yeast is Saccharomyces distaticus.In another more preferred aspect, the yeast is Saccharomyces uvarum.

In a preferred aspect, the bacterium is a Bacillus. In a more preferredaspect, the bacterium is Bacillus coagulans. In another preferredaspect, the bacterium is a Clostridium. In another more preferredaspect, the bacterium is Clostridium acetobutylicum. In another morepreferred aspect, the bacterium is Clostridium phytofermentans. Inanother more preferred aspect, the bacterium is Clostridiumthermocellum. In another more preferred aspect, the bacterium isGeobacillus sp. In another more preferred aspect, the bacterium is aThermoanaerobacter. In another more preferred aspect, the bacterium isThermoanaerobacter saccharolyticum. In another preferred aspect, thebacterium is a Zymomonas. In another more preferred aspect, thebacterium is Zymomonas mobilis.

Commercially available yeast suitable for ethanol production include,e.g., BIOFERM™ AFT and XR (NABC—North American Bioproducts Corporation,GA, USA), ETHANOL RED™ yeast (Fermentis/Lesaffre, USA), FALI™(Fleischmann's Yeast, USA), FERMIOL™ (DSM Specialties), GERT STRAND™(Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC™ fresh yeast(Ethanol Technology, WI, USA).

In a preferred aspect, the fermenting microorganism has been geneticallymodified to provide the ability to ferment pentose sugars, such asxylose utilizing, arabinose utilizing, and xylose and arabinoseco-utilizing microorganisms.

The cloning of heterologous genes into various fermenting microorganismshas led to the construction of organisms capable of converting hexosesand pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloningand improving the expression of Pichia stipitis xylose reductase gene inSaccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Hoet al., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation bySaccharomyces cerevisiae, Appl. Micro biol. Biotechnol. 38: 776-783;Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiaestrains overexpressing the TKL1 and TALI genes encoding the pentosephosphate pathway enzymes transketolase and transaldolase, Appl.Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimalmetabolic engineering of Saccharomyces cerevisiae for efficientanaerobic xylose fermentation: a proof of principle, FEMS Yeast Research4: 655-664; Beall et al., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering ofbacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhanget al., 1995, Metabolic engineering of a pentose metabolism pathway inethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al.,1996, Development of an arabinose-fermenting Zymomonas mobilis strain bymetabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470;WO 2003/062430, xylose isomerase).

In a preferred aspect, the genetically modified fermenting microorganismis Candida sonorensis. In another preferred aspect, the geneticallymodified fermenting microorganism is Escherichia coli. In anotherpreferred aspect, the genetically modified fermenting microorganism isKlebsiella oxytoca. In another preferred aspect, the geneticallymodified fermenting microorganism is Kluyveromyces mancianus. In anotherpreferred aspect, the genetically modified fermenting microorganism isSaccharomyces cerevisiae. In another preferred aspect, the geneticallymodified fermenting microorganism is Zymomonas mobilis.

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The fermenting microorganism is typically added to the degradedcellulosic material or hydrolysate and the fermentation is performed forabout 8 to about 96 hours, e.g., about 24 to about 60 hours. Thetemperature is typically between about 26° C. to about 60° C., e.g.,about 32° C. or 50° C., and about pH 3 to about pH 8, e.g., pH 4-5, 6,or 7.

In one aspect, the yeast and/or another microorganism are applied to thedegraded cellulosic material and the fermentation is performed for about12 to about 96 hours, such as typically 24-60 hours. In another aspect,the temperature is preferably between about 20° C. to about 60° C.,e.g., about 25° C. to about 50° C., about 32° C. to about 50° C., orabout 32° C. to about 50° C., and the pH is generally from about pH 3 toabout pH 7, e.g., about pH 4 to about pH 7. However, some fermentingorganisms, e.g., bacteria, have higher fermentation temperature optima.Yeast or another microorganism is preferably applied in amounts ofapproximately 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰,especially approximately 2×10⁸ viable cell count per ml of fermentationbroth. Further guidance in respect of using yeast for fermentation canbe found in, e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P.Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom1999), which is hereby incorporated by reference.

A fermentation stimulator can be used in combination with any of theprocesses described herein to further improve the fermentation process,and in particular, the performance of the fermenting microorganism, suchas, rate enhancement and ethanol yield. A “fermentation stimulator”refers to stimulators for growth of the fermenting microorganisms, inparticular, yeast. Preferred fermentation stimulators for growth includevitamins and minerals. Examples of vitamins include multivitamins,biotin, pantothenate, nicotinic acid, meso-inositol, thiamine,pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and VitaminsA, B, C, D, and E. See, for example, Alfenore et al., Improving ethanolproduction and viability of Saccharomyces cerevisiae by a vitaminfeeding strategy during fed-batch process, Springer-Verlag (2002), whichis hereby incorporated by reference. Examples of minerals includeminerals and mineral salts that can supply nutrients comprising P, K,Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation Products:

A fermentation product can be any substance derived from thefermentation. The fermentation product can be, without limitation, analcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol,methanol, ethylene glycol, 1,3-propanediol [propylene glycol],butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane,hexane, heptane, octane, nonane, decane, undecane, and dodecane), acycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, andcyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); anamino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine,and threonine); a gas (e.g., methane, hydrogen (H₂), carbon dioxide(CO₂), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); anorganic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbicacid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaricacid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid,3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonicacid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, andxylonic acid); and polyketide. The fermentation product can also beprotein as a high value product.

In a preferred aspect, the fermentation product is an alcohol. It willbe understood that the term “alcohol” encompasses a substance thatcontains one or more hydroxyl moieties. In a more preferred aspect, thealcohol is n-butanol. In another more preferred aspect, the alcohol isisobutanol. In another more preferred aspect, the alcohol is ethanol. Inanother more preferred aspect, the alcohol is methanol. In another morepreferred aspect, the alcohol is arabinitol. In another more preferredaspect, the alcohol is butanediol. In another more preferred aspect, thealcohol is ethylene glycol. In another more preferred aspect, thealcohol is glycerin. In another more preferred aspect, the alcohol isglycerol. In another more preferred aspect, the alcohol is1,3-propanediol. In another more preferred aspect, the alcohol issorbitol. In another more preferred aspect, the alcohol is xylitol. See,for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999,Ethanol production from renewable resources, in Advances in BiochemicalEngineering/Biotechnology, Scheper, T., ed., Springer-Verlag BerlinHeidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002,The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processes forfermentative production of xylitol a sugar substitute, ProcessBiochemistry 30(2): 117-124; Ezeji et al., 2003, Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in siturecovery by gas stripping, World Journal of Microbiology andBiotechnology 19 (6): 595-603.

In another preferred aspect, the fermentation product is an alkane. Thealkane can be an unbranched or a branched alkane. In another morepreferred aspect, the alkane is pentane. In another more preferredaspect, the alkane is hexane. In another more preferred aspect, thealkane is heptane. In another more preferred aspect, the alkane isoctane. In another more preferred aspect, the alkane is nonane. Inanother more preferred aspect, the alkane is decane. In another morepreferred aspect, the alkane is undecane. In another more preferredaspect, the alkane is dodecane.

In another preferred aspect, the fermentation product is a cycloalkane.In another more preferred aspect, the cycloalkane is cyclopentane. Inanother more preferred aspect, the cycloalkane is cyclohexane. Inanother more preferred aspect, the cycloalkane is cycloheptane. Inanother more preferred aspect, the cycloalkane is cyclooctane.

In another preferred aspect, the fermentation product is an alkene. Thealkene can be an unbranched or a branched alkene. In another morepreferred aspect, the alkene is pentene. In another more preferredaspect, the alkene is hexene. In another more preferred aspect, thealkene is heptene. In another more preferred aspect, the alkene isoctene.

In another preferred aspect, the fermentation product is an amino acid.In another more preferred aspect, the organic acid is aspartic acid. Inanother more preferred aspect, the amino acid is glutamic acid. Inanother more preferred aspect, the amino acid is glycine. In anothermore preferred aspect, the amino acid is lysine. In another morepreferred aspect, the amino acid is serine. In another more preferredaspect, the amino acid is threonine. See, for example, Richard andMargaritis, 2004, Empirical modeling of batch fermentation kinetics forpoly(glutamic acid) production and other microbial biopolymers,Biotechnology and Bioengineering 87(4): 501-515.

In another preferred aspect, the fermentation product is a gas. Inanother more preferred aspect, the gas is methane. In another morepreferred aspect, the gas is H₂. In another more preferred aspect, thegas is CO₂. In another more preferred aspect, the gas is CO. See, forexample, Kataoka et al., 1997, Studies on hydrogen production bycontinuous culture system of hydrogen-producing anaerobic bacteria,Water Science and Technology 36(6-7): 41-47; and Gunaseelan, 1997,Biomass and Bioenergy, 13(1-2): 83-114, 1997, Anaerobic digestion ofbiomass for methane production: A review.

In another preferred aspect, the fermentation product is isoprene.

In another preferred aspect, the fermentation product is a ketone. Itwill be understood that the term “ketone” encompasses a substance thatcontains one or more ketone moieties. In another more preferred aspect,the ketone is acetone. See, for example, Qureshi and Blaschek, 2003,supra.

In another preferred aspect, the fermentation product is an organicacid. In another more preferred aspect, the organic acid is acetic acid.In another more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen and Lee, 1997, Membrane-mediated extractivefermentation for lactic acid production from cellulosic biomass, Appl.Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the fermentation product is polyketide.

Recovery.

The fermentation product(s) can be optionally recovered from thefermentation medium using any method known in the art including, but notlimited to, chromatography, electrophoretic procedures, differentialsolubility, distillation, or extraction. For example, alcohol isseparated from the fermented cellulosic material and purified byconventional methods of distillation. Ethanol with a purity of up toabout 96 vol. % can be obtained, which can be used as, for example, fuelethanol, drinking ethanol, i.e., potable neutral spirits, or industrialethanol.

Signal Peptide

The present invention also relates to an isolated polynucleotideencoding a signal peptide comprising or consisting of amino acids 1 to19 of SEQ ID NO: 2. The polynucleotide may further comprise a geneencoding a protein, which is operably linked to the signal peptide. Theprotein is preferably foreign to the signal peptide. In one aspect, thepolynucleotide for the signal peptide is nucleotides 1 to 57 of SEQ IDNO: 1.

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides.

The present invention also relates to methods of producing a protein,comprising: (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andpolypeptides. The term “protein” also encompasses two or morepolypeptides combined to form the encoded product. The proteins alsoinclude hybrid polypeptides and fused polypeptides.

Preferably, the protein is a hormone, enzyme, receptor or portionthereof, antibody or portion thereof, or reporter. For example, theprotein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an alpha-galactosidase, alpha-glucosidase,aminopeptidase, amylase, beta-galactosidase, beta-glucosidase,beta-xylosidase, carbohydrase, carboxypeptidase, catalase,cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, endoglucanase, esterase,glucoamylase, invertase, laccase, lipase, mannosidase, mutanase,oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Media

COVE N medium was composed of 218 g of sorbitol, 50 ml of COVE saltsolution, 10 g of dextrose, 2.02 g of KNO₃, 25 g of agar, and deionizedwater to 1 liter.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCl, 76 gof KH₂PO₄, 50 ml of COVE trace metals solution, and deionized water to 1liter.

COVE trace metals solution was composed of 0.04 g Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄. H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionized water to 1 liter.

DAP4C-1 medium was composed of 11 g of MgSO₄.7H₂O, 1 g KH₂PO₄, 2 g ofcitric acid, monohydrate, 20 g of dextrose, 10 g of maltose, 6 g ofK₃PO₄.3H₂O, 0.5 g of yeast extract, 0.5 ml of trace metals solution, 1ml of Pluronic, and deionized water to 1 liter. The medium was portionedout to flasks, adding 250 mg CaCO₃ to each 150 ml portion. The mediumwas sterilized in an autoclave. After cooling the following was added to150 ml of medium: 3.5 ml of filter sterilized 50% w/v (NH₄)₂HPO₄, and5.0 ml of filter sterilized 20% lactic acid.

DAP4C-1 medium trace metals solution was composed of 6.8 g of ZnCl₂, 2.5g of CuSO₄.5H₂O, 0.24 g of NiCl₂.6H₂O, 13.9 g of FeSO₄.7H₂O, 8.45 g ofMnSO₄.H₂O, 3 g of citric acid monohydrate, and deionized water to 1liter.

MDU2BP medium was composed per liter of 45 g of maltose, 1 g ofMgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂SO₄, 12 g of KH₂PO₄, 7 g of yeastextract, 2 g of urea, 0.5 ml of AMG trace metals solution, and deionizedwater to 1 liter; pH 5.0.

AMG trace metals solution was composed of 14.3 g of ZnSO₄.7H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.7H₂O, 3 g of citric acid, and deionized water to 1 liter.

MY25 medium was composed per liter of 25 g of maltodextrin, 2 g ofMgSO₄.7H₂O, 10 g of KH₂PO₄, 2 g of citric acid, 2 g of K₂SO₄, 2 g ofurea, 10 g of yeast extract, 1.5 ml of AMG trace metals solution, anddeionized water to 1 liter; adjusted to pH 6.

SY50 medium was composed of 50 g of sucrose, 2 g of MgSO₄0.7H2O, 10 g ofKH₂PO₄, anhydrous, 2 g of K₂SO₄, 2 g of citric acid, 10 g of yeastextract, 2 g of urea, 0.5 g of CaCl₂.2H₂O, and 0.5 g of 200×AMG tracemetals solution, and deionized water to 1 liter; pH 6.0.

200×AMG trace metals solution was composed of 3 g of citric acid, 14.3 gof ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, and deionized water to 1 liter.

Example 1: PCR Amplification of a Cellobiohydrolase Gene from theGenomic DNA of Talaromyces byssochlamydoides CBS 413.71

A cellobiohydrolase encoding gene was PCR amplified from the genomic DNAof Talaromyces byssochlamydoides CBS 413.71 in a two step process.First, a central fragment of the gene was PCR amplified using degenerateprimers designed to match two conserved regions of sequence in genescoding for known Family GH6 cellobiohydrolase enzymes. Afteramplification of the internal fragment the sequence of the fragment wasdetermined and used to design gene-specific primers for gene walking inboth the 5′ and 3′ directions to obtain the entire coding sequence.

The internal gene fragment was PCR amplified using the degenerateprimers 859 and 860 shown below in a touch-down PCR protocol in whichthe initial annealing temperature of 67° C. was decreased by 1° C. ineach successive cycle for a total of 10 cycles, until an annealingtemperature of 57° C. was reached. The PCR amplification was thencompleted with an additional 29 cycles utilizing a 57° C. annealingtemperature.

Primer 859: (SEQ ID NO: 3) TKCCYGAYCGYGAYTGYGC Primer 860:(SEQ ID NO: 4) TCRCCACCKGGCTTKAYCCA

The amplification was performed using a REDDYMIX™ PCR Master Mix (ABgeneLtd, Epsom, UK). The amplification reaction was composed of 1 μl of T.byssochlamydoides CBS 413.71 genomic DNA as template, 50 μmol each ofprimers 859 and 860, and 12.5 μl of REDDYMIX™ PCR Master Mix in a finalvolume of 25 μl. T. byssochlamydoides genomic DNA was was extracted fromfresh mycelium using a FastDNA® SPIN protocol (Qbiogene, Inc., Carlsbad,Calif., USA). The amplification was performed in a thermal cyclerprogrammed for an initial template denaturation step at 94° C. for 2minutes; 11 cycles with denaturing at 94° C. for 45 seconds, annealingat 67° C. for 45 seconds with a decrease of 1° C. for each subsequentcycle, and elongation at 72° C. for 1 minute; and 29 cycles withdenaturing at 94° C. for 45 seconds, annealing at 57° C. for 45 seconds,and extension at 72° C. for 1 minute. A final elongation was made at 72°C. for 7 minutes.

The reaction products were resolved by 1% agarose gel electrophoresiswhere a PCR product band of approximately 700-800 bp was observed. Theband was excised from the gel and the DNA purified using an ILLUSTRA™GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, LittleChalfont, UK). The purified PCR fragment was cloned into vectorpCR®2.1-TOPO® (Invitrogen, Life Technologies, Carlsbad, Calif., USA)using a TOPO® TA CLONING® Kit (Invitrogen, Life Technologies, Carlsbad,Calif., USA) according to the manufacturer's instructions and thentransformed into TOP10 Chemically Competent E. coli cells (Invitrogen,Life Technologies, Carlsbad, Calif., USA) according to themanufacturer's instructions.

The sequence of the PCR product was determined directly with primers 859and 860, and by sequencing 4 individual clones of the PCR product withM13 forward and M13 reverse vector primers shown below.

M13 forward: (SEQ ID NO: 5) TGTAAAACGACGGCCAGT M13 reverse:(SEQ ID NO: 6) AGCGGATAACAATTTCACACAGGThe sequence was compared to known sequences using the BLAST search tool(Altschul et al., 1990, J. Mol. Biol. 215: 403-410) and confirmed to besimilar to known cellobiohydrolase encoding genes.

The partial sequence of the Talaromyces byssochlamydoidescellobiohydrolase encoding gene was used to design gene specific primers934, 935, 1044, and 1045 shown below to enable gene walking from bothends of the sequence.

Primer 934: (SEQ ID NO: 7) AGAGTCTCGTCTCAGTACATG Primer 935:(SEQ ID NO: 8) CGAATACGTCACCAGCCAC Primer 1044: (SEQ ID NO: 9)AATTGCTGAGCTGTTTCAGC Primer 1045: (SEQ ID NO: 10) TGACTGGTGCAACGTGATCG

Gene walking was performed using a DNA Walking SPEEDUP™ Premix Kit(Seegene, Seoul, Korea) based on the manufacturer's protocol with someminor differences. Only the first two sets of PCR reactions described inthe protocol were utilized, which included one initial set ofamplifications with a gene-specific primer and four different returnprimers and one set of nested reactions with a second gene-specificprimer. Half of the recommended reaction volumes were used for the firstset of reactions.

For walking in the 5′ direction, the first set of PCR reactions wasperformed with the gene-specific primer 934. After amplification, thereactions were diluted with 150 μl of water, and 5 μl of the dilutionwas used as template in the second nested set of PCR reactions withgene-specific primer 935. The second amplifications were performed asdescribed by the DNA Walking SPEEDUP™ Premix Kit protocol with a 58° C.annealing temperature. Reaction products were resolved by 1% agarose gelelectrophoresis, where a faint single band was observed of approximately1000 bp in one of the four nested reactions. The 1000 bp fragment wasre-amplified twice, first by repeating the nested PCR reaction using 1μl of the reaction including the 1000 bp product as template. Thereaction products were resolved by 1% agarose gel electrophoresis, and asecond re-amplification was made from this reaction by removing a smallpiece of the 1000 bp band from the gel with a pipette tip, which wasused as template in a PCR reaction under the same conditions. Thereaction products were resolved by 1% agarose gel electrophoresis using40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer andthe 1000 bp band was excised from the gel and the DNA purified using anILLUSTRA™ GFX™ PCR DNA and Gel Band Purification Kit. The sequence ofthe PCR product was determined using primer 935.

For walking in the 3′ direction, the first set of PCR reactions wasperformed with the gene-specific primer 1044. After amplification, thereactions were diluted with 150 μl of water, and 5 μl of the dilutionwas used as template in the second nested set of PCR reactions withgene-specific primer 1045. The second amplifications were performed asdescribed by the DNA Walking SPEEDUP™ Premix Kit protocol with a 56° C.annealing temperature. The reaction products were purified from the PCRreaction components using an ILLUSTRA™ GFX™ PCR DNA and Gel BandPurification Kit and concentrated by eluting in 10 μl of elution buffersupplied with the Kit. The products were analyzed by first cloning 4 μlof each purified PCR reaction directly into pCR®2.1-TOPO® using a TOPOTA CLONING® Kit reaction and transforming the TOPO TA CLONING® Kitreactions into TOP10 Chemically Competent E. coli cells (Invitrogen,Life Technologies, Carlsbad, Calif., USA) according to themanufacturer's instructions. The clones obtained were screened forinserts by restriction digestion, and those containing inserts weresequenced with M13 forward (SEQ ID NO: 5) and M13 reverse (SEQ ID NO: 6)vector primers. Four individual clones each of approximately 800 bpprovided the 3′ sequence for the T. byssochlamydoides cellobiohydrolaseencoding gene. All sequences were assembled into a single contig.

The genomic DNA sequence and deduced amino acid sequence of theTalaromyces byssochlamydoides cellobiohydrolase encoding sequence areshown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The genomic DNAsequence of 1789 bp (including the stop codon) contains 7 intronslocated at nucleotides 80 to 131, 201 to 253, 540 to 592, 847 to 897,1036 to 1095, 1354 to 1443, and 1686 to 1744 of SEQ ID NO: 1. Thegenomic DNA fragment encodes a polypeptide of 456 amino acids. The % G+Ccontent of the mature polypeptide coding sequence is 56%. Using theSignalP software program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 19 residues was predicted. The predictedmature protein contains 437 amino acids with a predicted molecular massof 46 kDa and an isoelectric point of 4.0. The protein contains acellulose binding module of the CBM1 type at the N terminus (amino acids20 to 56 of SEQ ID NO: 2). The catalytic domain is amino acids 98 to456.

A comparative alignment of mature cellobiohydrolase amino acidsequences, without the signal peptides, was determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of EMBOSS with gapopen penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62matrix. The alignment showed that the deduced amino acid sequence of theTalaromyces byssochlamydoides cellobiohydrolase (mature polypeptide)shares 84% identity (excluding gaps) to the deduced amino acid sequenceof a cellobiohydrolase from Talaromyces emersonii (UNIPROT: Q8NIB5).

Example 2: Cloning of the Talaromyces byssochlamydoidesCellobiohydrolase Encoding Gene into an Aspergillus Expression Vector

The T. byssochlamydoides cellobiohydrolase encoding gene was cloned intothe Aspergillus expression vector pMStr57 (WO 2004/032648) by PCRamplifying the protein coding sequence from genomic DNA with twosynthetic oligonucleotide primers shown below. Vector pMStr57 containssequences for selection and propagation in E. coli, and selection andexpression in Aspergillus. Selection in Aspergillus is facilitated bythe amdS gene of Aspergillus nidulans, which allows the use of acetamideas a sole nitrogen source. Expression in Aspergillus is mediated by amodified neutral amylase II (NA2) promoter from Aspergillus niger whichis fused to the 5′ leader sequence of the triose phosphate isomerase(tpi) encoding-gene from Aspergillus nidulans, and the terminator fromthe amyloglucosidase-encoding gene from Aspergillus niger.

Primer 1167: (SEQ ID NO: 11) ACACAACTGGGGATCCTCACCATGCGAAATATTCTTGPrimer 1168: (SEQ ID NO: 12) CCCTCTAGATCTCGAGCTAGAATGACGGATTGGCGTT

The amplification was performed using IPROOF™ High Fidelity 2× MasterMix (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) following themanufacturer's instructions. The amplification reaction was composed ofT. byssochlamydoides CBS 413.71 genomic DNA as a template, 25 pmol eachof primers 1167 and 1168, and 25 μl of IPROOF™ High Fidelity 2× MasterMix in a final volume of 50 μl. The amplification was performed in athermal cycler programmed for an initial template denaturation step at98° C. for 2 minutes; 5 cycles each with denaturing at 98° C. for 10seconds, annealing at 65° C. for 10 seconds, and elongation at 72° C.for 1 minute; and 30 cycles each with denaturing at 98° C. for 10seconds, and combined annealing extension at 72° C. for 1 minute. Afinal elongation was made at 72° C. for 10 minutes.

A PCR product of approximately 2000 bp was separated from residualreaction components using a GFX® PCR DNA and Gel Band Purification Kitaccording to the manufacturer's instructions. The purified PCR fragmentwas sequenced, and the sequence agreed completely with the sequence ofSEQ. ID NO. 1.

The PCR fragment was cloned into Barn HI and Xho I digested pMStr57using an IN-FUSION™ Dry-Down PCR Cloning Kit (Clontech Laboratories,Inc., Mountain View, Calif., USA) according to the manufacturer'sinstructions. The Talaromyces byssochlamydoides cellobiohydrolaseencoding DNA of the resulting Aspergillus expression construct,pMStr215, was sequenced and the sequence agreed completely with thesequence of SEQ ID NO: 1.

Example 3: Expression of the Talaromyces byssochlamydoidesCellobiohydrolase Encoding Gene in Aspergillus oryzae MT3568

The fungal expression host Aspergillus oryzae strain MT3568 wastransformed with pMStr215 according to Christensen et al., 1988,Biotechnology 6, 1419-1422 and WO 2004/032648. Aspergillus oryzae MT3568is an amdS (acetamidase) gene disrupted derivative of Aspergillus oryzaeJaL355 (WO 2002/40694) in which pyrG auxotrophy was restored in theprocess of knocking out the A. oryzae amdS gene. Eight transformantswere cultured for 4 days at 30° C. in 750 μl of DAP2C-1 medium (WO2004/032648). Samples were monitored by SDS-PAGE using E-PAGE 48 8% gelswith SeeBlue Plus2 molecular weight standards (Invitrogen, LifeTechnologies, Carlsbad, Calif., USA) according to the manufacturer'sinstructions. The gel was stained with INSTANTBLUE™ (Expedeon ProteinSolutions, Cambridge, UK). Six transformants produced a novel proteinband of approximately 55 kDa.

Two of these transformants, designated Aspergillus oryzae MStr390 andMStr391, were isolated twice by dilution streaking conidia on selectivemedium (amdS) containing 0.01% TRITON® X-100 to limit colony size.

Example 4: Shake Flask Cultures of the Recombinant Aspergillus oryzaeMStr391 for the Production of the Talaromyces byssochlamydoidesCellobiohydrolase

Spores from two confluent COVE N slants of Aspergillus oryzae MStr391,designated EXP03666, were collected with a solution of 0.01% TWEEN® 20and used to inoculate 10 shake flasks each containing 150 ml of DAP4C-1medium. The flasks were incubated at 30° C. for 4 days with constantshaking at 200 rpm. Fungal mycelia and spores were removed at harvestingby first filtering the fermentation broth through a sandwich of 3 glassmicrofiber filters with increasing particle retention sizes of 1.6 μm,1.2 μm and 0.7 μm, and then filtering through a 0.45 μm groundwaterfilter.

Filtered broth was added to 1.8 M ammonium sulphate and adjusted to pH5.6. After filtration on a 0.22 μm PES filter (Nalge Nunc International,Rochester, N.Y., USA), the filtrate was loaded onto a Phenyl Sepharose™6 Fast Flow column (high sub) (GE Healthcare, Piscataway, N.J., USA)equilibrated with 1.8 M ammonium sulphate pH 5.6, and bound proteinswere eluted with 25 mM HEPES pH 7.0. The fractions were pooled andapplied to a SEPHADEX™ G-25 (medium) (GE Healthcare, Piscataway, N.J.,USA) column equilibrated in 50 mM HEPES pH 7.0. The fractions wereapplied to a SOURCE™ 15Q (GE Healthcare, Piscataway, N.J., USA) columnequilibrated in 50 mM HEPES pH 7.0, and bound proteins were eluted witha linear gradient from 0-1000 mM sodium chloride. Protein concentrationwas determined using a Microplate BCA™ Protein Assay Kit (Thermo FischerScientific, Waltham, Mass., USA) in which bovine serum albumin was usedas a protein standard.

Example 5: Pretreated Corn Stover Hydrolysis Assay

Corn stover was pretreated at the U.S. Department of Energy NationalRenewable Energy Laboratory (NREL) using 1.4 wt % sulfuric acid at 165°C. and 107 psi for 8 minutes. The water-insoluble solids in thepretreated corn stover (PCS) contained 56.5% cellulose, 4.6%hemicellulose, and 28.4% lignin. Cellulose and hemicellulose weredetermined by a twostage sulfuric acid hydrolysis with subsequentanalysis of sugars by high performance liquid chromatography using NRELStandard Analytical Procedure #002. Lignin was determinedgravimetrically after hydrolyzing the cellulose and hemicellulosefractions with sulfuric acid using NREL Standard Analytical Procedure#003.

Unmilled, unwashed PCS (whole slurry PCS) was prepared by adjusting thepH of the PCS to 5.0 by addition of 10 M NaOH with extensive mixing, andthen autoclaving for 20 minutes at 120° C. The dry weight of the wholeslurry PCS was 29%. Milled unwashed PCS (dry weight 32.35%) was preparedby milling whole slurry PCS in a Cosmos ICMG 40 wet multi-utilitygrinder (EssEmm Corporation, Tamil Nadu, India). Milled washed PCS (dryweight 32.35%) was prepared in the same manner, with subsequent washingwith deionized water and decanting off the supernatant fractionrepeatedly.

The hydrolysis of PCS was conducted using 2.2 ml deep-well plates(Axygen, Union City, Calif., USA) in a total reaction volume of 1.0 ml.The hydrolysis was performed with 50 mg of insoluble PCS solids per mlof 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganese sulfateand various protein loadings of various enzyme compositions (expressedas mg protein per gram of cellulose). Enzyme compositions were preparedand then added simultaneously to all wells in a volume ranging from 50μl to 200 μl, for a final volume of 1 ml in each reaction. The plate wasthen sealed using an ALPS300™ plate heat sealer (Abgene, Epsom, UnitedKingdom), mixed thoroughly, and incubated at a specific temperature for72 hours. All experiments reported were performed in triplicate.

Following hydrolysis, samples were filtered using a 0.45 μm MULTISCREEN®96-well filter plate (Millipore, Bedford, Mass., USA) and filtratesanalyzed for sugar content as described below. When not usedimmediately, filtered aliquots were frozen at −20° C. The sugarconcentrations of samples diluted in 0.005 M H₂SO₄ were measured using a4.6×250 mm AMINEXO HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,Calif., USA) by elution with 0.05% w/w benzoic acid-0.005 M H₂SO₄ at 65°C. at a flow rate of 0.6 ml per minute, and quantitation by integrationof the glucose, cellobiose, and xylose signals from refractive indexdetection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, SantaClara, Calif., USA) calibrated by pure sugar samples. The resultantglucose and cellobiose equivalents were used to calculate the percentageof cellulose conversion for each reaction.

Glucose, cellobiose, and xylose were measured individually. Measuredsugar concentrations were adjusted for the appropriate dilution factor.The net concentrations of enzymatically-produced sugars from unwashedPCS were determined by adjusting the measured sugar concentrations forcorresponding background sugar concentrations in unwashed PCS at zerotime point. All HPLC data processing was performed using MICROSOFTEXCEL™ software (Microsoft, Richland, Wash., USA).

The degree of cellulose conversion to glucose was calculated using thefollowing equation: % conversion=(glucose concentration/glucoseconcentration in a limit digest)×100. To calculate total conversion theglucose and cellobiose values were combined. Cellobiose concentrationwas multiplied by 1.053 in order to convert to glucose equivalents andadded to the glucose concentration. The degree of total celluloseconversion was calculated using the following equation: %conversion=([glucose concentration+1.053×(cellobioseconcentration)]/[(glucose concentration+1.053×(cellobiose concentration)in a limit digest])×100. The 1.053 factor for cellobiose takes intoaccount the increase in mass when cellobiose is converted to glucose. Inorder to calculate % conversion, a 100% conversion point was set basedon a cellulase control (50-100 mg of Trichoderma reesei cellulase pergram cellulose), and all values were divided by this number and thenmultiplied by 100. Triplicate data points were averaged and standarddeviation was calculated.

Example 6: Preparation of Aspergillus fumigatus NN055679 Cel7ACellobiohydrolase I

A tfasty search (Pearson et al., 1997, Genomics 46:24-36) of theAspergillus fumigatus partial genome sequence (The Institute for GenomicResearch, Rockville, Md.) was performed using as query a Ce17cellobiohydrolase protein sequence from Trichoderma reesei (AccessionNo. P00725). Several genes were identified as putative Family GH7homologs based upon a high degree of similarity to the query sequence atthe amino acid level. One genomic region with significant identity tothe query sequence was chosen for further study, and the correspondinggene was named cel7A.

Two synthetic oligonucleotide primers shown below were designed to PCRamplify an Aspergillus fumigatus NN055679 cel7A cellobiohydrolase 1 gene(SEQ ID NO: 13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acidsequence]) from genomic DNA of Aspergillus fumigatus prepared asdescribed in WO 2005/047499.

Forward primer: (SEQ ID NO: 15) 5′-gggcATGCTGGCCTCCACCTTCTCC-3′Reverse primer: (SEQ ID NO: 16) 5′-gggttaattaaCTACAGGCACTGAGAGTAA-3′

Upper case letters represent the coding sequence. The remainder of thesequence provides restriction endonuclease sites for Sph I and Pac I inthe forward and reverse sequences, respectively. Using these primers,the Aspergillus fumigatus cel7A gene was amplified using standard PCRmethods and the reaction product isolated by 1% agarose gelelectrophoresis using TAE buffer and purified using a QIAQUICK® GelExtraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions.

The fragment was digested with Sph I and Pac I and ligated into theexpression vector pAILo2 also digested with Sph I and Pac I according tostandard procedures. The ligation products were transformed into E. coliXL10 SOLOPACK® cells (Stratagene, La Jolla, Calif., USA) according tothe manufacturer's instructions. An E. coli transformant containing aplasmid of the correct size was detected by restriction digestion andplasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia,Calif., USA). DNA sequencing of the insert gene from this plasmid wasperformed with an Applied Biosystems Model 377 XL Automated DNASequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif.,USA) using dye-terminator chemistry (Giesecke et al., 1992, Journal ofVirology Methods 38: 47-60) and primer walking strategy. Nucleotidesequence data were scrutinized for quality and all sequences werecompared to each other with assistance of PHRED/PHRAP software(University of Washington, Seattle, Wash., USA). The nucleotide sequencewas shown to match the genomic sequence determined by TIGR (SEQ ID NO:13 [DNA sequence] and SEQ ID NO: 14 [deduced amino acid sequence]). Theresulting plasmid was named pEJG93.

Aspergillus oryzae JaL250 (WO 99/61651) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra andtransformed with 5 μg of pEJG93 (as well as pAILo2 as a vector control)was used to transform Aspergillus oryzae JaL250. The transformation ofyielded about 100 transformants. Ten transformants were isolated toindividual PDA plates.

Confluent PDA plates of five of the ten transformants were washed with 5ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of MDU2BPmedium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm.Five days after incubation, 0.5 μl of supernatant from each culture wasanalyzed using 8-16% Tris-Glycine SDS-PAGE gels (Invitrogen, Carlsbad,Calif., USA) according to the manufacturer's instructions. SDS-PAGEprofiles of the cultures showed that one of the transformants had amajor band of approximately 70 kDa. This transformant was namedAspergillus oryzae JaL250EJG93.

Five hundred ml of shake flask medium were added to a 2800 ml shakeflask. The shake flask medium was composed of 45 g of maltose, 2 g ofK₂HPO₄, 12 g of KH₂PO₄, 1 g of NaCl, 1 g of MgSO₄.7H₂O, 7 g of yeastextract, 2 g of urea, 0.5 ml of trace elements solution, and deionizedwater to 1 liter. The trace elements solution was composed of 13.8 g ofFeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄. H₂O, 2.5 g ofCuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 3 g of citric acid, and deionized waterto 1 liter. Two shake flasks were inoculated with a suspension of a PDAplate of Aspergillus oryzae JaL250EJG93 with 0.01% TWEEN® 80 andincubated at 34° C. on an orbital shaker at 200 rpm for 120 hours. Thebroth was filtered using a 0.7 μm Whatman glass filter GF/F (Whatman,Piscataway, N.J., USA) followed by a 0.22 μm EXPRESS™ Plus Membrane(Millipore, Bedford, Mass., USA).

Filtered broth was concentrated and buffer exchanged with 20 mM Tris-HClpH 8.5 using a tangential flow concentrator (Pall Filtron, Northborough,Mass., USA) equipped with a 10 kDa polyethersulfone membrane (PallFiltron, Northborough, Mass., USA). Protein concentration was determinedusing a Microplate BCA™ Protein Assay Kit in which bovine serum albuminwas used as a protein standard.

Example 7: Preparation of Thermoascus aurantiacus CGMCC 0670 Cel5AEndoglucanase II

Thermoascus aurantiacus CGMCC 0670 cDNA encoding a Cel5A endoglucanaseII (SEQ ID NO: 17 [DNA sequence] and SEQ ID NO: 18 [deduced amino acidsequence]) was cloned according to the following procedure. The T.aurantiacus strain was grown in 80 ml of CBH1 medium (2.5% AVICEL®, 0.5%glucose, 0.14% (NH₄)₂SO₄) in 500 ml Erlenmeyer baffled flasks at 45° C.for 3 days with shaking at 165 rpm. Mycelia were harvested bycentrifugation at 7000 rpm for 30 minutes and stored at −80° C. beforeuse for RNA extraction. RNA was isolated from 100 mg of mycelia using aRNEASY® Plant Mini Kit (QIAGEN Inc., Valencia, Calif., USA).

The cDNA for the Thermoascus aurantiacus endoglucanase was isolated byRT PCR using a 3′ RACE system and a 5′ RACE System (Invitrogen, LifeTechnologies, Carlsbad, Calif., USA) and primers BG025-1, BG025-2,BG025-3, and BG025-4 shown below to the N-terminal amino acids.

Primer BG025-1: (SEQ ID NO: 19) 5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)GC(T/C/A/G)GAATT-3′ Primer BG025-2: (SEQ ID NO: 20)5′-AA(T/C)GA(A/G)TC(T/C/A/G)GG(T/C/A/G)G C(T/C/A/G)GAGTT-3′Primer BG025-3: (SEQ ID NO: 21) 5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/C/A/G)GAATT-3′ Primer BG025-4: (SEQ ID NO: 22)5′-AA(T/C)GA(A/G)AG(T/C)GG(T/C/A/G)GC(T/ C/A/G)GAGTT-3′

The RT PCR products were ligated into plasmid pGEMO-T using a pGEMO-TVector System (Promega, Madison, Wis., USA) and transformed into E. colistrain JM109. A single clone harboring a plasmid named pBGC1009containing the endoglucanase cDNA was isolated.

PCR primers were designed to amplify the cDNA encoding the T aurantiacusendoglucanase from plasmid pBGC1009. Restriction enzyme sites Bsp HI andPac I were incorporated for in-frame cloning into Aspergillus oryzaeexpression plasmid pBM120a (WO 2006/039541).

Primer 996261: (SEQ ID NO: 23) 5′-GATCTCATGAAGCTCGGCTCTCTCGT-3′       BspHI Primer 996167: (SEQ ID NO: 24)5′-TTAATTAATCAAAGATACGGAGTCAAAATAGG-3′      PacI

The fragment of interest was amplified by PCR using an EXPAND™ HighFidelity PCR System. The PCR amplification reaction mixture contained 1μl of 0.09 μg/μl pBGC1009, 1 μl of primer 996261 (50 pmol/μl), 1 μl ofprimer 996167 (50 pmol/μl), 5 μl of 10×PCR buffer with 15 mM MgCl₂, 1 μlof dNTP mix (10 mM each), 37.25 μl of water, and 0.75 μl (3.5 U/μl) ofDNA polymerase mix. An EPPENDORF® MASTERCYCLER® thermocycler (EppendorfScientific, Inc., Westbury, N.Y., USA) was used to amplify the fragmentprogrammed for 1 cycle at 94° C. for 2 minutes; 10 cycles each at 94° C.for 15 seconds, 55° C. for 30 seconds, 72° C. for 1.5 minutes; 15 cycleseach at 94° C. for 15 seconds, 55° C. for 30 seconds, and 72° C. for 1.5minutes plus 5 second elongation at each successive cycle; 1 cycle at72° C. for 7 minutes; and a 4° C. hold.

The 1008 bp PCR product was purified by 1% agarose gel electrophoresisusing TAE buffer, excised from the gel, and purified using a QIAQUICK®Gel Purification Kit (QIAGEN Inc., Valencia, Calif., USA). The purifiedproduct was ligated directly into pCR®2.1-TOPO® according to themanufacturer's instructions. The resulting plasmid was named pBM124a.

Plasmid pBM124a was digested with Bsp HI and Pac I, purified by 1%agarose gel electrophoresis using TAE buffer, excised from the gel, andpurified using a QIAQUICK® Gel Purification Kit. The plasmid fragmentwas ligated to the vector pBM120a, which was digested with Nco I and PacI. The resulting expression plasmid was designated pBM123a. PlasmidpBM123a contains a duplicate NA2-TPI promoter driving expression of theThermoascus aurantiacus endoglucanase cDNA clone, the AMG terminator,and amdS as a selectable marker.

Aspergillus oryzae BECh2 (WO 2000/139322) protoplasts were preparedaccording to the method of Christensen et al., 1988, supra andtransformed with 6 μg of pBM123a. Primary transformants were selected onCOVE plates for 5 days. Transformants were spore purified twice prior toshake flask analysis.

Spores of the transformants were inoculated into 25 ml of MY25 medium in125 ml shake flasks. The cultures were incubated at 34° C., 200 rpm on aplatform shaker for five days. On day 3 and day 5, culture supernatantswere harvested and clarified by centrifugation to remove mycelia. Twentymicroliters of supernatant from three transformants were analyzed usinga CRITERION® stain-free, 10-20% gradient SDS-PAGE gel (Bio-RadLaboratories, Inc., Hercules, Calif., USA) according to themanufacturer's instructions. SDS-PAGE profiles of the cultures showedthat all transformants had a new major band of approximately 32 kDa. Onetransformant was chosen and named A. oryzae EXP00858.

Plastic, non-baffled 500 ml shake flasks containing 100 ml of SY50medium were inoculated with 0.1 ml of a spore stock of A. oryzaeEXP00858, and incubated at 34° C., 200 rpm for 24 hours to produce aseed culture. Fifty ml of the seed culture was inoculated into a 2 literfermentation tank containing 2 liters of medium composed per liter of0.5 g of pluronic acid, 30 g of sucrose, 2 g of MgSO₄7H₂O, 2 g ofanhydrous KH₂PO₄, 1 g of citric acid, 2 g of (NH₄)₂SO₄, 1 g of K₂50₄, 20g of yeast extract, and 0.5 g of 200×AMG trace metals solution, pH 5.0.The fermentation was fed with a maltose feed. The pH was controlledusing 5 N H₃PO₄ and 15% NH₄OH and maintained at 5.0 and then raised to5.25. Temperature was maintained 34.0° C.+/−1.0° C. Agitation was 1000rpm. Airflow was 1.0 vvm.

A 200 ml volume of cell-free supernatant was diluted to 1 liter withdeionized water. The pH was adjusted to 8 and the sample filtersterilized using a 0.22 μm polyethersulphone (PES) filter. The filtersterilized sample was loaded onto a 250 ml Q SEPHAROSE™ Fast Flow column(GE Healthcare, Piscataway, N.J., USA) pre-equilibrated with 25 mM TrispH 8. The enzyme was eluted from the column with a 0 to 1 M NaOHgradient in the same buffer. The fractions containing beta-glucosidaseactivity were pooled (400 ml) and the enzyme concentration calculatedfrom the theoretic extinction coefficient and the absorbance of thesample at 280 nm.

Example 8: Preparation of Thermoascus aurantiacus CGMCC 0583 GH61APolypeptide Having Cellulolytic Enhancing Activity

Thermoascus aurantiacus CGMCC 0583 GH61A polypeptide having cellulolyticenhancing activity (SEQ ID NO: 25 [DNA sequence] and SEQ ID NO: 26[deduced amino acid sequence]) was recombinantly prepared according toWO 2005/074656 using Aspergillus olyzae JaL250 as a host. Therecombinantly produced Thermoascus aurantiacus GH61A polypeptide wasfirst concentrated by ultrafiltration using a 10 kDa membrane, bufferexchanged into 20 mM Tris-HCl pH 8.0, and then purified using a 100 ml QSEPHAROSE® Big Beads column (GE Healthcare, Piscataway, N.J., USA) with600 ml of a 0-600 mM NaCl linear gradient in the same buffer. Fractionsof 10 ml were collected and pooled based on SDS-PAGE.

The pooled fractions (90 ml) were then further purified using a 20 mlMONO Q® column (GE Healthcare, Piscataway, N.J., USA) with 500 ml of a0-500 mM NaCl linear gradient in the same buffer. Fractions of 6 ml werecollected and pooled based on SDS-PAGE. The pooled fractions (24 ml)were concentrated by ultrafiltration using a 10 kDa membrane, andchromatographed using a 320 ml SUPERDEX® 75 SEC column (GE Healthcare,Piscataway, N.J., USA) with isocratic elution of approximately 1.3liters of 150 mM NaCl-20 mM Tris-HCl pH 8.0. Fractions of 20 ml werecollected and pooled based on SDS-PAGE. Protein concentration wasdetermined using a Microplate BCA™ Protein Assay Kit in which bovineserum albumin was used as a protein standard.

Example 9: Preparation of Aspergillus fumigatus NN055679 Cel3Abeta-glucosidase

Aspergillus fumigatus NN055679 Cel3A beta-glucosidase (SEQ ID NO: 27[DNA sequence] and SEQ ID NO: 28 [deduced amino acid sequence]) wasrecombinantly prepared according to WO 2005/047499 using Trichodermareesei RutC30 as a host.

Filtered broth was concentrated and buffer exchanged using a tangentialflow concentrator equipped with a 10 kDa polyethersulfone membrane with20 mM Tris-HCl pH 8.5. The sample was loaded onto a Q SEPHAROSE® HighPerformance column (GE Healthcare, Piscataway, N.J., USA) equilibratedin 20 mM Tris pH 8.5, and bound proteins were eluted with a lineargradient from 0-600 mM sodium chloride. The fractions were concentratedand loaded onto a SUPERDEX® 75 HR 26/60 column GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris-150 mM sodiumchloride pH 8.5. Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit in which bovine serum albumin was used as aprotein standard.

Example 10: Preparation of Aspergillus fumigatus NN055679 GH10 Xylanase

Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (SEQ ID NO: 29 [DNAsequence] and SEQ ID NO: 30 [deduced amino acid sequence]) was preparedrecombinantly according to WO 2006/078256 using Aspergillus oryzae BECh2as a host.

The filtered broth was desalted and buffer-exchanged into 20 mM Tris-150mM NaCl pH 8.5 using a HIPREPO 26/10 Desalting Column (GE Healthcare,Piscataway, N.J., USA) according to the manufacturer's instructions.Protein concentration was determined using a Microplate BCA™ ProteinAssay Kit with bovine serum albumin as a protein standard.

Example 11: Effect of the Talaromyces byssochlamydoides Family GH6Cellobiohydrolase in the Hydrolysis of Milled Unwashed PCS at 50-65° C.by a High-Temperature Enzyme Composition

The Talaromyces byssochlamydoides Family GH6 cellobiohydrolase wasevaluated in a high-temperature enzyme composition at 50° C., 55° C.,60° C., and 65° C. using milled unwashed PCS as a substrate. Thehigh-temperature enzyme composition included 40% Aspergillus fumigatusCel7A cellobiohydrolase I, 25% Talaromyces byssochlamydoides Family GH6polypeptide cellobiohydrolase, 10% Thermoascus aurantiacus Cel5Aendoglucanase II, 15% Thermoascus aurantiacus GH61A polypeptide havingcellulolytic enhancing activity, 5% Aspergillus fumigatus Cel3Abeta-glucosidase, and 5% Aspergillus fumigatus GH10 xylanase (xyn3). Thehigh-temperature enzyme composition was added to PCS hydrolysisreactions at 3.0 mg total protein per g cellulose, and the hydrolysisresults were compared with the results for a similar high-temperatureenzyme composition without the T. byssochlamydoides GH6cellobiohydrolase (2.25 mg protein per g cellulose).

The assay was performed as described in Example 5. The 1 ml reactionswith milled unwashed PCS (5% insoluble solids) were conducted for 72hours in 50 mM sodium acetate pH 5.0 buffer containing 1 mM manganesesulfate. All reactions were performed in triplicate and involved singlemixing at the beginning of hydrolysis.

The results shown in FIG. 1 demonstrated that at 50° C., 55° C., 60° C.,and 65° C. the high-temperature enzyme composition that included 25% T.byssochlamydoides Family GH6 cellobiohydrolase significantlyoutperformed the enzyme composition without the T. byssochlamydoidescellobiohydrolase.

Example 12: Preparation of Aspergillus fumigatus Cellobiohydrolase

Aspergillus fumigatus NN055679 cellobiohydrolase (SEQ ID NO: 31 [DNAsequence] and SEQ ID NO: 32 [deduced amino acid sequence]) was preparedrecombinantly in Aspergillus olyzae as described in WO 2011/057140. Thefiltered broth of Aspergillus fumigatus GH6A cellobiohydrolase wasbuffer exchanged into 20 mM Tris pH 8.0 using a 400 ml SEPHADEX™ G-25column (GE Healthcare, United Kingdom) according to the manufacturer'sinstructions. The fractions were pooled and adjusted to 1.2 M ammoniumsulphate-20 mM Tris pH 8.0. The equilibrated protein was loaded onto aPHENYL SEPHAROSE™ 6 Fast Flow column (high sub) (GE Healthcare,Piscataway, N.J., USA) equilibrated in 20 mM Tris pH 8.0 with 1.2 Mammonium sulphate, and bound proteins were eluted with 20 mM Tris pH 8.0with no ammonium sulphate. The fractions were pooled. Proteinconcentration was determined using a Microplate BCA™ Protein Assay Kitwith bovine serum albumin as a protein standard.

Example 13: Preparation of Myceliophthora thermophila CBS 202.75 GH6ACellobiohydrolase

Myceliophthora thermophila CBS 117.65 GH6A cellobiohydrolase (SEQ ID NO:33 [DNA sequence] and SEQ ID NO: 34 [deduced amino acid sequence]) wasprepared recombinantly in Aspergillus olyzae as described in WO2011/057140. The filtered broth of Myceliophthora thermophila GH6Acellobiohydrolase was buffer exchanged into 20 mM Tris pH 8.0 using a400 ml Sephadex G-25 column (GE Healthcare, United Kingdom) according tothe manufacturer's instructions. The buffer exchanged broth was adjustedto 20 mM Tris-HCl pH 8.0 with 1.2 M ammonium sulfate and applied to aPHENYL SEPHAROSE™ 6 Fast Flow column (high sub) (GE Healthcare,Piscataway, N.J., USA) equilibrated with 20 mM Tris pH 8.0 with 1.2 Mammonium sulfate. The bound proteins were eluted with 20 mM Tris pH 8.0with no ammonium sulfate and fractions were pooled. The pooled fractionswere concentrated and buffer exchanged into 20 mM Tris pH 8.0 using a 10kDa MWCO Amicon Ultra centrifuge concentrator (Millipore, Bedford,Mass., USA). Protein concentration was determined using a MicroplateBCA™ Protein Assay Kit with bovine serum albumin as a protein standard.

Example 14: Evaluation of Three Cellobiohydrolases on Milled Washed PCSat 50-65° C.

Three cellobiohydrolases (Talaromyces byssochlamydoides GH6cellobiohydrolase, Aspergillus fumigatus GH6A cellobiohydrolase, andMyceliophthora thermophila GH6A cellobiohydrolase) were evaluated at 1mg protein per g cellulose at 50° C., 55° C., 60° C., and 65° C. usingmilled washed PCS as a substrate with 1 mg protein per g cellulose ofAspergillus fumigatus beta-glucosidase.

The assay was performed as described in Example 5. The 1 ml reactionswith milled washed PCS (5% insoluble solids) were conducted for 72 hoursin 50 mM sodium acetate pH 4.0, 4.5, and 5.0 buffer containing 1 mMmanganese sulfate. All reactions were performed in triplicate andinvolved single mixing at the beginning of hydrolysis.

The results shown in FIG. 2 demonstrated that at 50° C., 55° C., 60° C.,and 65° C. and pH 4.0, 4.5, and 5.0 the Talaromyces byssochlamydoidesGH6 cellobiohydrolase (Tb6) yielded a higher conversion of milled washedPCS than Aspergillus fumigatus GH6A cellobiohydrolase (Af6A) orMyceliophthora thermophila GH6A cellobiohydrolase (Mt6A) when comparedunder the same conditions of temperature and pH.

Example 15: Determination of Td by Differential Scanning Calorimetry

The thermostability of the Talaromyces byssochlamydoides GH6cellobiohydrolase was determined by Differential Scanning calorimetry(DSC) using a VP-Capillary Differential Scanning calorimeter (MicroCalInc., Piscataway, N.J., USA). The thermal denaturation temperature, Td(° C.), was taken as the top of denaturation peak (major endothermicpeak) in thermograms (Cp vs. T) obtained after heating enzyme solutionin 50 mM sodium acetate pH 5.0 at a constant programmed heating rate of200 K/hr. Sample- and reference-solutions (approximately 0.2 ml) wereloaded into the calorimeter (reference: buffer without enzyme) fromstorage conditions at 10° C. and thermally pre-equilibrated for 20minutes at 20° C. prior to DSC scanning from 20° C. to 110° C. The Tdfor Talaromyces byssochlamydoides GH6 cellobiohydrolase was determinedto be 74° C.+/−1° C. at pH 5.0.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection (NRRL), Northern Regional Research Center, 1815University Street, Peoria, Ill., USA, and given the following accessionnumber:

Deposit Accession Number Date of Deposit E. coli (pAJ227) NRRL B-50474Mar. 1, 2011

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by foreign patent laws to be entitledthereto. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The present invention is further described by the following numberedparagraphs:

[1] An isolated polypeptide having cellobiohydrolase activity, selectedfrom the group consisting of: (a) a polypeptide having at least 85%,e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the mature polypeptide of SEQ ID NO: 2; (b) apolypeptide encoded by a polynucleotide that hybridizes under very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii); (c) a polypeptide encoded by a polynucleotidehaving at least 85%, e.g., at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% sequence identity to the mature polypeptide codingsequence of SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant ofthe mature polypeptide of SEQ ID NO: 2 comprising a substitution,deletion, and/or insertion at one or more positions; and (e) a fragmentof the polypeptide of (a), (b), (c), or (d) that has cellobiohydrolaseactivity.

[2] The polypeptide of paragraph 1, having at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99% or 100% sequence identity to themature polypeptide of SEQ ID NO: 2.

[3] The polypeptide of paragraph 1 or 2, which is encoded by apolynucleotide that hybridizes under very high stringency conditionswith (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii)the cDNA sequence thereof, or (iii) the full-length complement of (i) or(ii).

[4] The polypeptide of any of paragraphs 1-3, which is encoded by apolynucleotide having at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the mature polypeptidecoding sequence of SEQ ID NO: 1 or the cDNA sequence thereof.

[5] The polypeptide of any of paragraphs 1-4, comprising or consistingof SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 2.

[6] The polypeptide of paragraph 5, wherein the mature polypeptide isamino acids 20 to 456 of SEQ ID NO: 2.

[7] The polypeptide of any of paragraphs 1-4, which is a variant of themature polypeptide of SEQ ID NO: 2 comprising a substitution, deletion,and/or insertion at one or more positions.

[8] The polypeptide of paragraph 1, which is a fragment of SEQ ID NO: 2,wherein the fragment has cellobiohydrolase activity.

[9] The polypeptide of paragraph 5, which is encoded by a polynucleotidewhich is identical to the polynucleotide contained in plasmid pAJ227which is contained in E. coli NRRL B-50474.

[10] The polypeptide of paragraph 5, which is identical to thepolypeptide encoded by the polynucleotide contained in plasmid pAJ227which is contained in E. coli NRRL B-50474.

[11] An isolated polypeptide comprising a catalytic domain selected fromthe group consisting of: (a) a catalytic domain having at least 90%,e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to amino acids 98 to 456 of SEQ ID NO: 2; (b) acatalytic domain encoded by a polynucleotide that hybridizes under veryhigh stringency conditions with (i) nucleotides 397 to 1786 of SEQ IDNO: 1, (ii) the cDNA sequence thereof, or (iii) the full-lengthcomplement of (i) or (ii); (c) a catalytic domain encoded by apolynucleotide having at least 90%, e.g., at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to nucleotides 397 to1786 of SEQ ID NO: 1 or the cDNA sequence thereof; (d) a variant ofamino acids 98 to 456 of SEQ ID NO: 2 comprising a substitution,deletion, and/or insertion at one or more positions; and (e) a fragmentof a catalytic domain of (a), (b), (c), or (d) that hascellobiohydrolase activity.

[12] The polypeptide of paragraph 11, further comprising a cellulosebinding domain.

[13] An isolated polypeptide comprising a cellulose binding domainoperably linked to a catalytic domain, wherein the cellulose bindingdomain is selected from the group consisting of: (a) a cellulose bindingdomain having at least 90%, e.g., at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to amino acids 20 to 56 ofSEQ ID NO: 2; (b) a cellulose binding domain encoded by a polynucleotidethat hybridizes under very high stringency conditions with (i)nucleotides 58 to 273 of SEQ ID NO: 1, (ii) the cDNA sequence thereof,or (iii) the full-length complement of (i) or (ii); (c) a cellulosebinding domain encoded by a polynucleotide having at least 90%, e.g., atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% sequenceidentity to nucleotides 58 to 273 of SEQ ID NO: 1 or the cDNA sequencethereof; (d) a variant of amino acids 20 to 56 of SEQ ID NO: 2comprising a substitution, deletion, and/or insertion at one or morepositions; and (e) a fragment of (a), (b), (c), or (d) that hascellulose binding activity.

[14] The polypeptide of paragraph 13, wherein the catalytic domain isobtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase, ortransferase, e.g., an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellobiohydrolase, cellobiohydrolase,cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase,deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolyticenzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase, xylanase, or beta-xylosidase.

[15] A composition comprising the polypeptide of any of paragraphs 1-14.

[16] An isolated polynucleotide encoding the polypeptide of any ofparagraphs 1-14.

[17] A nucleic acid construct or expression vector comprising thepolynucleotide of paragraph 16 operably linked to one or more controlsequences that direct the production of the polypeptide in an expressionhost.

[18] A recombinant host cell comprising the polynucleotide of paragraph16 operably linked to one or more control sequences that direct theproduction of the polypeptide.

[19] A method of producing the polypeptide of any of paragraphs 1-14,comprising: (a) cultivating a cell, which in its wild-type form producesthe polypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[20] A method of producing a polypeptide having cellobiohydrolaseactivity, comprising: (a) cultivating the host cell of paragraph 18under conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

[21] A transgenic plant, plant part or plant cell transformed with apolynucleotide encoding the polypeptide of any of paragraphs 1-14.

[22] A method of producing a polypeptide having cellobiohydrolaseactivity, comprising: (a) cultivating the transgenic plant or plant cellof paragraph 21 under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide.

[23] A method of producing a mutant of a parent cell, comprisinginactivating a polynucleotide encoding the polypeptide of any ofparagraphs 1-14, which results in the mutant producing less of thepolypeptide than the parent cell.

[24] A mutant cell produced by the method of paragraph 23.

[25] The mutant cell of paragraph 24, further comprising a gene encodinga native or heterologous protein.

[26] A method of producing a protein, comprising: (a) cultivating themutant cell of paragraph 24 or 25 under conditions conducive forproduction of the protein; and (b) recovering the protein.

[27] A double-stranded inhibitory RNA (dsRNA) molecule comprising asubsequence of the polynucleotide of paragraph 16, wherein optionallythe dsRNA is an siRNA or an miRNA molecule.

[28] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph27, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or moreduplex nucleotides in length.

[29] A method of inhibiting the expression of a polypeptide havingcellobiohydrolase activity in a cell, comprising administering to thecell or expressing in the cell the double-stranded inhibitory RNA(dsRNA) molecule of paragraph 27 or 28.

[30] A cell produced by the method of paragraph 29.

[31] The cell of paragraph 30, further comprising a gene encoding anative or heterologous protein.

[32] A method of producing a protein, comprising: (a) cultivating thecell of paragraph 30 or 31 under conditions conducive for production ofthe protein; and (b) recovering the protein.

[33] An isolated polynucleotide encoding a signal peptide comprising orconsisting of amino acids 1 to 19 of SEQ ID NO: 2.

[34] A nucleic acid construct or expression vector comprising a geneencoding a protein operably linked to the polynucleotide of paragraph33, wherein the gene is foreign to the polynucleotide encoding thesignal peptide.

[35] A recombinant host cell comprising a gene encoding a proteinoperably linked to the polynucleotide of paragraph 33, wherein the geneis foreign to the polynucleotide encoding the signal peptide.

[36] A method of producing a protein, comprising: (a) cultivating arecombinant host cell comprising a gene encoding a protein operablylinked to the polynucleotide of paragraph 33, wherein the gene isforeign to the polynucleotide encoding the signal peptide, underconditions conducive for production of the protein; and (b) recoveringthe protein.

[37] A method for degrading or converting a cellulosic material,comprising: treating the cellulosic material with an enzyme compositionin the presence of the polypeptide having cellobiohydrolase activity ofany of paragraphs 1-14.

[38] The method of paragraph 37, wherein the cellulosic material ispretreated.

[39] The method of paragraph 37 or 38, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[40] The method of paragraph 39, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[41] The method of paragraph 39, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[42] The method of any of paragraphs 37-41, further comprisingrecovering the degraded cellulosic material.

[43] The method of paragraph 42, wherein the degraded cellulosicmaterial is a sugar.

[44] The method of paragraph 43, wherein the sugar is selected from thegroup consisting of glucose, xylose, mannose, galactose, and arabinose.

[45] A method for producing a fermentation product, comprising: (a)saccharifying a cellulosic material with an enzyme composition in thepresence of the polypeptide having cellobiohydrolase activity of any ofparagraphs 1-14; (b) fermenting the saccharified cellulosic materialwith one or more fermenting microorganisms to produce the fermentationproduct; and (c) recovering the fermentation product from thefermentation.

[46] The method of paragraph 45, wherein the cellulosic material ispretreated.

[47] The method of paragraph 45 or 46, wherein the enzyme compositioncomprises one or more enzymes selected from the group consisting of acellulase, a GH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[48] The method of paragraph 47, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[49] The method of paragraph 47, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[50] The method of any of paragraphs 45-49, wherein steps (a) and (b)are performed simultaneously in a simultaneous saccharification andfermentation.

[51] The method of any of paragraphs 45-50, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[52] A method of fermenting a cellulosic material, comprising:fermenting the cellulosic material with one or more fermentingmicroorganisms, wherein the cellulosic material is saccharified with anenzyme composition in the presence of the polypeptide havingcellobiohydrolase activity of any of paragraphs 1-14.

[53] The method of paragraph 52, wherein the fermenting of thecellulosic material produces a fermentation product.

[54] The method of paragraph 53, further comprising recovering thefermentation product from the fermentation.

[55] The method of any of paragraphs 52-54, wherein the cellulosicmaterial is pretreated before saccharification.

[56] The method of any of paragraphs 52-55, wherein the enzymecomposition comprises one or more enzymes selected from the groupconsisting of a cellulase, a GH61 polypeptide having cellulolyticenhancing activity, a hemicellulase, an esterase, an expansin, alaccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease,and a swollenin.

[57] The method of paragraph 56, wherein the cellulase is one or moreenzymes selected from the group consisting of an endoglucanase, acellobiohydrolase, and a beta-glucosidase.

[58] The method of paragraph 56, wherein the hemicellulase is one ormore enzymes selected from the group consisting of a xylanase, anacetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, axylosidase, and a glucuronidase.

[59] The method of any of paragraphs 53-58, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.

[60] A whole broth formulation or cell culture composition comprising apolypeptide of any of paragraphs 1-14.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

What is claimed is:
 1. A method for degrading a cellulosic material,comprising: treating the cellulosic material with an enzyme compositioncomprising a polypeptide having cellobiohydrolase activity, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 95% sequence identity to amino acids 20 to 456of SEQ ID NO:
 2. 2. The method of claim 1, further comprising recoveringthe degraded cellulosic material.
 3. A method for producing afermentation product, comprising: (a) saccharifying a cellulosicmaterial with an enzyme composition comprising a polypeptide havingcellobiohydrolase activity, wherein the polypeptide havingcellobiohydrolase activity comprises an amino acid sequence having atleast 95% sequence identity to amino acids 20 to 456 of SEQ ID NO: 2;(b) fermenting the saccarified cellulosic material with one or morefermenting microorganisms to produce the fermentation product; and (c)recovering the fermentation product from the fermentation.
 4. A methodof fermenting a cellulosic material, comprising: fermenting thecellulosic material with one or more fermenting microorganisms, whereinthe cellulosic material is saccharified with an enzyme compositioncomprising a polypeptide having cellobiohydrolase activity, wherein thepolypeptide having cellobiohydrolase activity comprises an amino acidsequence having at least 95% sequence identity to amino acids 20 to 456of SEQ ID NO:
 2. 5. The method of claim 4, wherein the fermenting of thecellulosic material produces a fermentation product.
 6. The method ofclaim 5, further comprising recovering the fermentation product from thefermentation.
 7. A whole broth formulation or cell culture compositioncomprising a polypeptide having cellobiohydralase activity, wherein thepolypeptide having cellobiohydralase activity comprises an amino acidsequence having at least 95% sequence identity to amino acids 20 to 456of SEQ ID NO:
 2. 8. The method of claim 1, wherein the polypeptidehaving cellobiohydrolase activity has at least 96% sequence identity toamino acids 20 to 456 of SEQ ID NO:
 2. 9. The method of claim 1, whereinthe polypeptide having cellobiohydrolase activity has at least 97%sequence identity to amino acids 20 to 456 of SEQ ID NO:
 2. 10. Themethod of claim 1, wherein the polypeptide having cellobiohydrolaseactivity has at least 98% sequence identity to amino acids 20 to 456 ofSEQ ID NO:
 2. 11. The method of claim 1, wherein the polypeptide havingcellobiohydrolase activity has at least 99% sequence identity to aminoacids 20 to 456 of SEQ ID NO:
 2. 12. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity comprises amino acids 20to 456 of SEQ ID NO:
 2. 13. The method of claim 1, wherein thepolypeptide having cellobiohydrolase activity is encoded by thepolynucleotide of SEQ ID NO: 1 which is identical to the polynucleotidecontained in plasmid pAJ227 which is contained in E. coli deposited withthe Agricultural Research Service Patent Culture Collection (NRRL) underaccession number B-50474.
 14. The method of claim 1, wherein thecellulosic material is pretreated.
 15. The method of claim 1, whereinthe enzyme composition further comprises one or more enzymes selectedfrom the group consisting of a cellulase, a GH61 polypeptide havingcellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 16. The method of claim 2, wherein thedegraded cellulosic material is a sugar.
 17. The method of claim 16,wherein the sugar is selected from the group consisting of glucose,xylose, mannose, galactose, and arabinose.
 18. The method of claim 3,wherein the polypeptide having cellobiohydrolase activity comprisesamino acids 20 to 456 of SEQ ID NO:
 2. 19. The method of claim 3,wherein the cellulosic material is pretreated.
 20. The method of claim3, wherein the enzyme composition further comprises one or more enzymesselected from the group consisting of a cellulase, a GH61 polypeptidehaving cellulolytic enhancing activity, a hemicellulase, an esterase, anexpansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, aprotease, and a swollenin.
 21. The method of claim 3, wherein steps (a)and (b) are performed simultaneously in a simultaneous saccharificationand fermentation.
 22. The method of claim 3, wherein the fermentationproduct is an alcohol, an alkane, a cycloalkane, an alkene, an aminoacid, a gas, isoprene, a ketone, an organic acid, or polyketide.
 23. Themethod of claim 4, wherein the polypeptide having cellobiohydrolaseactivity comprises amino acids 20 to 456 of SEQ ID NO:
 2. 24. The methodof claim 4, wherein the cellulosic material is pretreated.
 25. Themethod of claim 4, wherein the enzyme composition further comprises oneor more enzymes selected from the group consisting of a cellulase, aGH61 polypeptide having cellulolytic enhancing activity, ahemicellulase, an esterase, an expansin, a laccase, a ligninolyticenzyme, a pectinase, a peroxidase, a protease, and a swollenin.
 26. Themethod of claim 5, wherein the fermentation product is an alcohol, analkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, aketone, an organic acid, or polyketide.